U.S. patent application number 13/817054 was filed with the patent office on 2013-11-14 for compositions and methods for delivering nucleic acid molecules and treating cancer.
This patent application is currently assigned to UNIVERSITY OF MEDICINE AND DENTISTRY OF NEW JERSEY. The applicant listed for this patent is Olga B. Garbuzenko, Tamara Minko, Lorna Rodriguez-Rodriguez, Vatsal Shah, Oleh Taratula. Invention is credited to Olga B. Garbuzenko, Tamara Minko, Lorna Rodriguez-Rodriguez, Vatsal Shah, Oleh Taratula.
Application Number | 20130302257 13/817054 |
Document ID | / |
Family ID | 45605653 |
Filed Date | 2013-11-14 |
United States Patent
Application |
20130302257 |
Kind Code |
A1 |
Minko; Tamara ; et
al. |
November 14, 2013 |
Compositions and Methods for Delivering Nucleic Acid Molecules and
Treating Cancer
Abstract
The present invention provides compositions and methods for the
delivery of nucleic acids to a cell. The present invention
additionally provides compositions and methods for the treatment of
a disease or disorder, particularly cancer.
Inventors: |
Minko; Tamara; (Somerset,
NJ) ; Rodriguez-Rodriguez; Lorna; (East Brunswick,
NJ) ; Garbuzenko; Olga B.; (Highland Park, NJ)
; Taratula; Oleh; (Corvallis, OR) ; Shah;
Vatsal; (New Brunswick, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Minko; Tamara
Rodriguez-Rodriguez; Lorna
Garbuzenko; Olga B.
Taratula; Oleh
Shah; Vatsal |
Somerset
East Brunswick
Highland Park
Corvallis
New Brunswick |
NJ
NJ
NJ
OR
NJ |
US
US
US
US
US |
|
|
Assignee: |
UNIVERSITY OF MEDICINE AND
DENTISTRY OF NEW JERSEY
Somerset
NJ
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
New Brunswick
NJ
|
Family ID: |
45605653 |
Appl. No.: |
13/817054 |
Filed: |
August 17, 2011 |
PCT Filed: |
August 17, 2011 |
PCT NO: |
PCT/US11/48078 |
371 Date: |
July 10, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61374413 |
Aug 17, 2010 |
|
|
|
Current U.S.
Class: |
424/9.361 ;
424/450; 424/9.3; 435/375; 514/34; 514/44A; 514/9.7; 530/350 |
Current CPC
Class: |
A61K 47/60 20170801;
C12N 2810/854 20130101; C12N 15/87 20130101; A61K 47/64 20170801;
A61K 47/59 20170801 |
Class at
Publication: |
424/9.361 ;
435/375; 514/44.A; 424/9.3; 424/450; 530/350; 514/34; 514/9.7 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Goverment Interests
[0002] This invention was made with government support under grant
Nos. CA138533, CA111766, and CA100098 awarded by the National
Institutes of Health, National Cancer Institute. The government has
rights in the invention.
Claims
1. A method for delivering an siRNA or antisense molecule to a
cell, said method comprising: a) forming a complex comprising at
least one siRNA or antisense molecule and a poly(propyleneimine)
(PPI) dendrimer, and b) contacting said cell with said complex.
2. The method of claim 1, wherein said PPI dendrimer is a
generation four (G4) or a generation five (G5) dendrimer.
3. The method of claim 2, wherein said PPI dendrimer is a G4
dendrimer.
4. The method of claim 1, wherein step b) comprises administering
said complex to a subject.
5. The method of claim 1, wherein said method is performed in
vitro.
6. The method of claim 1, wherein said PPI dendrimer is linked to
at least one cancer targeting ligand.
7. The method of claim 6, wherein said cancer targeting ligand is
linked to said PPI dendrimer via a polyethylene glycol
molecule.
8. The method of claim 6, wherein said cancer targeting ligand is
luteinizing hormone-releasing hormone (LHRH) or an analog
thereof.
9. The method of claim 8, wherein said cancer targeting ligand
comprises SEQ ID NO: 3.
10. The method of claim 1, wherein said complex further comprises
at least one chemotherapeutic agent.
11. The method of claim 10, wherein said complex further comprises
at least two chemotherapeutic agents with different mechanisms of
action.
12. The method of claim 10, wherein said chemotherapeutic agent is
selected from the group consisting of doxorubicin, cisplatin,
paclitaxel, camptothecin, and topotecan.
13. The method of claim 1, wherein said complex further comprises
an imaging agent.
14. The method of claim 13, wherein said imaging agent is a
magnetic resonance imaging (MRI) contrast agent.
15. The method of claim 14, wherein said MRI contrast agent is
superparamagnetic iron oxide (SPIO).
16. The method of claim 1, wherein said PPI dendrimer is complexed
with siRNA.
17. The method of claim 1, wherein said siRNA or antisense molecule
are inhibitors of cellular drug resistance.
18. The method of claim 17, wherein said inhibitors of cellular
drug resistance include an inhibitor of pump resistance and/or an
inhibitor of nonpump resistance.
19. The method of claim 18, wherein said inhibitor of pump
resistance is an inhibitor of Multi-Drug Resistance (MDR)
associated protein.
20. The method of claim 19, wherein said Multi-Drug Resistance
(MDR) associated protein is multidrug resistance protein (MRP) or
P-glycoprotein encoded by the multidrug resistance 1 (MDR1)
gene.
21. The method of claim 18, wherein said inhibitor of nonpump
resistance is an inhibitor of BCL2 or CD44.
22. A method of treating cancer in a patient in need thereof, said
method comprising administering to said patient at least two
chemotherapeutic agents with different mechanisms of action and at
least two inhibitors of cellular drug resistance.
23. The method of claim 22, wherein said chemotherapeutic agents
include doxorubicin and cisplatin.
24. The method of claim 22, wherein said inhibitors of cellular
drug resistance include an inhibitor of pump resistance and an
inhibitor of nonpump resistance.
25. The method of claim 24, wherein said inhibitor of pump
resistance is an inhibitor of Multi-Drug Resistance (MDR)
associated protein.
26. The method of claim 25, wherein said Multi-Drug Resistance
(MDR) associated protein is multidrug resistance protein (MRP) or
P-glycoprotein encoded by the multidrug resistance 1 (MDR1)
gene.
27. The method of claim 22, wherein said inhibitor is an antisense
molecule or a siRNA molecule.
28. The method of claim 24, wherein said inhibitor of nonpump
resistance is an inhibitor of BCL2 or CD44.
29. The method of claim 28, wherein said inhibitor is an antisense
molecule or a siRNA molecule.
30. The method of claim 22, wherein said chemotherapeutic agents
and said inhibitors of cellular drug resistance are contained
within a drug delivery system comprising at least one liposome
and/or a poly(propyleneimine) (PPI) dendrimer as a nanocarrier.
31. The method of claim 30, wherein said drug delivery system
comprises at least one cancer targeting ligand.
32. The method of claim 31, wherein said targeting ligand is
Luteinizing Hormone-Releasing Hormone (LHRH) or an analog
thereof.
33. The method of claim 32, wherein the LHRH analog comprises SEQ
ID NO: 3.
34. The method of claim 30, wherein said PPI dendrimer is a
generation four (G4) or a generation five (G5) dendrimer.
35. The method of claim 22, wherein said cancer is ovarian
cancer.
36. A compound comprising a polyamidoamine (PAMAM) dendrimer,
polyethylene glycol (PEG), and poly-L-lysine.
37. The compound of claim 36, wherein said PAMAM is linked to said
PEG and said PEG is linked to said poly-L-lysine.
38. The compound of claim 36, wherein said PAMAM is acetylated.
39. The compound of claim 36, wherein said PAMAM dendrimer is a G4
dendrimer.
40. The compound of claim 36, wherein the average molecular weight
of said PEG is about 2,000 to 5,000 Da.
41. The compound of claim 36, wherein the average molecular weight
of said poly-L-lysine is about 10,000 to about 20,000 Da.
42. A method of delivering an siRNA or antisense molecule to a
cell, said method comprising: a) forming a complex comprising at
least one siRNA or antisense molecule and the compound of claim 36,
and b) contacting said cell with said complex.
43. A composition comprising at least one liposome or dendrimer
comprising at least two chemotherapeutic agents with different
mechanisms of action and at least two inhibitors of cellular drug
resistance, and at least one pharmaceutically acceptable
carrier.
44. The composition of claim 43, wherein said chemotherapeutic
agents include doxorubicin and cisplatin.
45. The composition of claim 43, wherein said inhibitors of
cellular drug resistance include an inhibitor of pump resistance
and inhibitor of nonpump resistance.
46. The composition of claim 45, wherein said inhibitor of pump
resistance is an inhibitor of Multi-Drug Resistance (MDR)
associated protein.
47. The composition of claim 46, wherein said Multi-Drug Resistance
(MDR) associated protein is multidrug resistance protein (MRP) or
P-glycoprotein encoded by the multidrug resistance 1 (MDR1)
gene.
48. The composition of claim 43, wherein said inhibitor is an
antisense molecule or a siRNA molecule.
49. The composition of claim 45, wherein said inhibitor of nonpump
resistance is an inhibitor of BCL2.
50. The composition of claim 49, wherein said inhibitor is an
antisense molecule or a siRNA molecule.
51. The composition of claim 43, wherein said liposome or dendrimer
further comprises at least one cancer targeting ligand.
52. The composition of claim 51, wherein said cancer targeting
ligand is luteinizing hormone-releasing hormone (LHRH) or an analog
thereof.
53. The composition of claim 52, wherein the LHRH analog is SEQ ID
NO: 3.
Description
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Patent Application No. 61/374,413,
filed Aug. 17, 2010. The foregoing application is incorporated by
reference herein.
FIELD OF THE INVENTION
[0003] The present invention relates generally to methods of
delivering a nucleic acid molecule to patient. The present
invention also relates to compositions and methods for the
treatment of cancer.
BACKGROUND OF THE INVENTION
[0004] Ovarian cancer is one of the most common causes of cancer
death from gynecologic malignancy in the industrialized world.
Standard treatment involves aggressive cytoreductive (debulking)
surgery followed by chemotherapy. Ovarian cancer may spread to the
lining of the abdominal cavity as intraperitoneal metastases
(carcinomatosis) and leads to ascites. In general, carcinomatosis
and ascites indicate a more advanced stage of the disease that
usually requires extensive high dose chemotherapy (Berkenblit et
al. (2005) J. Reprod. Med., 50:426-438; Kawaguchi et al. (2005)
Curr. Drug Targets Cardiovasc. Haematol. Disord., 5:39-64).
Furthermore, tumor cells from malignant ascites are more invasive
and resistant to chemotherapy when compared with primary ovarian
tumors (Lane et al. (2010) J. Ovarian Res., 3:1; Tang et al. (2010)
Neoplasia 12, 128-138; Veatch et al. (1994) Int. J. Cancer
58:393-399). The precise mechanisms underlying the formation of
ascites in ovarian cancer are unknown. However, it is known that
the success of chemotherapeutic treatment of primary ovarian cancer
and especially tumor cells growing in ascitic fluid is limited by
the intrinsic and acquired resistance of cancer cells to
chemotherapy (Lane et al. (2010) J. Ovarian Res., 3:1; Tang et al.
(2010) Neoplasia 12, 128-138; Veatch et al. (1994) Int. J. Cancer
58:393-399; Li et al. (2009) BMC Cancer 9:323). Such resistance
requires the use of multiple chemotherapeutic agents thus
increasing the rate of severe adverse side effects of therapy on
healthy organs and tissues.
[0005] The main mechanisms of multidrug resistance are common to
most cancers and include "pump" and "nonpump" resistance (Liu et
al. (1998) Gynecol. Oncol., 70:398-403; Minko et al. (2004) Curr.
Drug Targets 5:389-406; Pakunlu et al. (2003) Pharm. Res.,
20:351-359; Pakunlu et al. (2004) Cancer Res., 64:6214-6224;
Krasznai et al. (2005) Anticancer Res., 25:1187-1192). Pump
resistance is caused by membrane transporters that pump out the
anticancer agents from cells, decreasing the intracellular drug
concentration and thereby the efficacy of the treatment. The main
mechanism of non-pump resistance is an activation of cellular
antiapoptotic defense. Effective treatment of advanced multidrug
resistant primary ovarian tumors and their intraperitoneal
metastases may be possible only by suppressing simultaneously at
least two main types of cellular resistance and by inducing cell
death using several anticancer agents with different mechanisms of
action. Such an objective can be best achieved if several
anticancer agents are simultaneously delivered specifically to the
tumor in combination with other active components that perform
different functions for enhancing cellular uptake and efficiency of
drugs in cancer cells, limiting adverse side effects, and
preventing the development of drug resistance and metastases.
SUMMARY OF THE INVENTION
[0006] In accordance with the instant invention, methods of
treating, preventing, or inhibiting cancer in a patient are
provided. In a particular embodiment, the method comprises
administering to a patient at least one chemotherapeutic agent
(particularly at least two chemotherapeutic agents with different
mechanisms of action) and at least one, particularly at least two,
inhibitors of cellular drug resistance (e.g., inhibitors of pump
resistance and/or inhibitors of nonpump resistance). In yet another
embodiment, the chemotherapeutic agents and the inhibitors of
cellular drug resistance are contained within a drug delivery
system (DDS) that utilizes a nanocarrier (e.g., a liposome or
dendrimer such as a poly(propyleneimine) (PPI) dendrimer). The DDS
may comprise at least one targeting ligand, particularly at least
one cancer targeting ligand, e.g., Luteinizing Hormone-Releasing
Hormone (LHRH) or an analog thereof.
[0007] According to another aspect of the instant invention,
methods of delivering a nucleic acid, particularly an siRNA or an
antisense molecule, to a cell are provided. In a particular method,
these methods are used in coordination with the above therapeutic
methods. In a particular embodiment, the methods of delivering an
siRNA or antisense molecule comprise A) forming a complex
comprising siRNA and an antisense molecule and a dendrimer (e.g., a
poly(propyleneimine) (PPI) dendrimer) and B) contacting a cell (in
vitro or in vivo) with the complex. In a particular embodiment, the
PPI dendrimer is a generation four (G4) or five (G5) dendrimer. In
yet another embodiment, the siRNA or antisense molecule is a
therapeutic molecule (e.g., an inhibitor of cellular drug
resistance) and the delivery of the siRNA or antisense molecule is
used to treat a disease or disorder (e.g., delivered to a subject).
In a particular embodiment, the disease or disorder is cancer. The
complex may further comprise a targeting ligand (e.g., cancer
targeting ligand) for directing the dendrimer to a specific cell
type (e.g., cancer cells). In a particular embodiment, the
targeting ligand is linked to the dendrimer via a polyethylene
glycol linker. In a particular embodiment, the targeting ligand is
luteinizing hormone-releasing hormone (LHRH) or an analog thereof.
The complex may further comprise at least one chemotherapeutic
agent. The complex may further comprise an imaging agent.
Compositions comprising at least one molecule of siRNA complexed
with the dendrimer and at least one pharmaceutically acceptable
carrier are provided.
[0008] According to another aspect of the instant invention,
compositions comprising a liposome or dendrimer comprising at least
one chemotherapeutic agents (particularly at least two
chemotherapeutic agents with different mechanisms of action), at
least one, particularly at least two, inhibitors of cellular drug
resistance (e.g., inhibitors of pump resistance and/or inhibitors
of nonpump resistance), and at least one pharmaceutically
acceptable carrier are provided. In another embodiment, the
liposome or dendrimer comprise all of the above components or part
of the above components. In certain embodiment, the siRNA molecules
are contained within the dendrimers.
[0009] According to another aspect of the instant invention, a
compound comprising a polyamidoamine (PAMAM) dendrimer,
polyethylene glycol (PEG), and poly-L-lysine is provided. The
compound is an effective carrier of nucleic acid molecules such as
siRNA and antisense molecules. Compositions comprising the
compound, at least one pharmaceutically acceptable carrier, and,
optionally, at least one siRNA or antisense molecule are also
encompassed by the instant invention. Methods of delivering an
siRNA or antisense molecule to a cell method comprising forming a
complex the above compound are also provided.
BRIEF DESCRIPTIONS OF THE DRAWING
[0010] FIG. 1A is a representative image of cells isolated from
human malignant ascites and transfected with luciferase or green
fluorescent protein. Cells isolated from human malignant ascites
were injected into the flanks of nude mice resulting in the
formation of solid primary tumor (FIG. 1B) and intraperitoneal
metastases (FIGS. 1C, 1D, and 1F). Typical bioluminescent (FIGS. 1B
and 1C, IVIS imaging system) and ultrasound (FIGS. 1D and 1F, Vevo
2100.RTM. imaging system) images of a live anesthetized mouse with
primary and metastatic tumors. FIG. 1E shows the expression of LHRH
receptors (LHRHR) in the plasma membrane of cells isolated from
human malignant ascites obtained from patients with ovarian
carcinoma. The cells were incubated with LHRH peptide labeled by
Rhodamine (red fluorescence). FIG. 1G shows the expression of LHRHR
and MDR1 gene encoding P-glycoprotein in cells isolated from human
malignant ascites. FIGS. 1H and 1I are representative images of gel
electrophoresis and quantitation of RT-PCR products of gene
encoding LHRHR. Means.+-.S.D. are shown. *P<0.05 when compared
with healthy tissues from the same patient. FIG. 1J is a schematic
of a multifunctional tumor-targeted liposomal delivery system.
[0011] FIG. 2 shows the histology of tumor and intraperitoneal
metastases. Treatment of aggressive subcutaneous tumor with
combination therapy (LHRH-Lip-DOX-BCL2-MDR1
ASO+LHRH-Lip-CIS-BCL2-MDR1 ASO) led to the significant changes in
histopathological pattern of the developed solid subcutaneous and
metastatic tumors. Upper panels: typical IVIS images of mice
bearing subcutaneous xenografts of human malignant ascites. The
tumor was accompanied by the development of intraperitoneal
metastases. Bottom panels: typical microscopy images of tumor
tissues and intraperitoneal metastases stained with
hematoxylin-eosin.
[0012] FIG. 3 demonstrates the gene expression in subcutaneous
tumors. FIG. 3A shows MDR1 gene expression and FIG. 3B shows BCL2
gene expression. Mice bearing xenografts of human malignant ascites
were treated 8 times within 30 days with the following substances:
(1) Saline (untreated control); (2) Liposomes (Lip); (3) LHRH; (4)
Doxorubicin (DOX); (5) Lip-DOX; (6) Lip-DOX-BCL2 ASO; (7)
Lip-DOX-MDR1 ASO; (8) Lip-DOX-BCL2-MDR1 ASO; (9)
LHRH-Lip-DOX-BCL2-MDR1 ASO; (10) Cisplatin (CIS); (11) Lip-CIS;
(12) Lip-CIS-BCL2 ASO; (13) Lip-CIS-MDR1 ASO; (14)
Lip-CIS-BCL2-MDR1 ASO; (15) LHRH-Lip-CIS-BCL2-MDR1 ASO; (16)
DOX+CIS; (17) Lip-DOX+Lip-CIS; (18) Lip-DOX-BCL2 ASO+Lip-CIS-BCL2
ASO; (19) Lip-DOX-MDR1 ASO+Lip-CIS-MDR1 ASO; (20) Lip-DOX-BCL2-MDR1
ASO+Lip-CIS-BCL2-MDR1 ASO; (21) LHRH-Lip-DOX-BCL2-MDR1
ASO+LHRH-Lip-CIS-BCL2-MDR1 ASO. Means.+-.S.D. are shown. *P<0.05
when compared with untreated control.
[0013] FIG. 4 shows the protein expression in subcutaneous tumors.
Typical images of tumor tissue sections stained with antibody
against P-glycoprotein (FIG. 4A), BCL2 (FIG. 4B) and Caspase 3
(FIG. 4C) proteins. High intensity of the color indicates high
protein concentration. Mice bearing xenografts of human malignant
ascites were treated 8 times within 30 days with the following
substances: (1) Saline (untreated control); (2) DOX+CIS; (3)
Lip-DOX; (4) Lip-CIS; (5) Lip-DOX+Lip-CIS; (6)
Lip-DOX-BCL2-MDR1-ASO; (7) LHRH-Lip-CIS-BCL2-MDR1-ASO; (8)
LHRH-Lip-DOX-BCL2-MDR1-ASO; (9) LHRH-Lip-CIS-BCL2-MDR1-ASO; (10)
LHRH-Lip-DOX-BCL2-MDR1-ASO+LHRH-Lip-CIS-BCL2-MDR1-ASO.
[0014] FIG. 5 provides graphs of the apoptosis induction in
subcutaneous tumors and intraperitoneal metastases. Mice bearing
xenografts of human malignant ascites were treated 8 times within
30 days with the following substances: (1) Saline (untreated
control); (2) Liposomes (Lip); (3) LHRH; (4) DOX; (5) Lip-DOX; (6)
Lip-DOX-BCL2 ASO; (7) Lip-DOX-MDR1 ASO; (8) Lip-DOX-BCL2-MDR1 ASO;
(9) LHRH-Lip-DOX-BCL2-MDR1 ASO; (10) CIS; (11) Lip-CIS; (12)
Lip-CIS-BCL2 ASO; (13) Lip-CIS-MDR1 ASO; (14) Lip-CIS-BCL2-MDR1
ASO; (15) LHRH-Lip-CIS-BCL2-MDR1 ASO; (16) DOX+CIS; (17)
Lip-DOX+Lip-CIS; (18) Lip-DOX-BCL2 ASO+Lip-CIS-BCL2 ASO; (19)
Lip-DOX-MDR1 ASO+Lip-CIS-MDR1 ASO; (20) Lip-DOX-BCL2-MDR1
ASO+Lip-CIS-BCL2-MDR1 ASO; (21) LHRH-Lip-DOX-BCL2-MDR1
ASO+LHRH-Lip-CIS-BCL2-MDR1 ASO. Means.+-.S.D. are shown.
[0015] FIG. 6 demonstrates the treatment with tumor-targeted
complex delivery systems containing two anticancer drugs with
different mechanisms of action and suppressors of pump and nonpump
cellular drug resistance substantially inhibits the growth of
subcutaneous tumor and prevents the development of intraperitoneal
metastases. Cancer cells were isolated from malignant ascites
obtained from patients with ovarian carcinoma and injected
subcutaneously into the flanks of nude mice. When the tumors
reached a size of about 0.3 cm.sup.3 (15-20 days after
transplantation), mice were treated maximum 8 times within 30 days
with substances indicated. FIG. 6A represents tumor growth during
the treatment and FIG. 6B represents the mass of intraperitoneal
metastases at the end of the treatment. Means.+-.S.D. are shown.
*P<0.05 when compared with untreated tumor (saline).
[0016] FIG. 7A provides a representative fluorescence spectra of
EtBr alone, EtBr after complexation with siRNA and EtBr after
displacement from siRNA by PPI G5 (N/P=2.4). FIG. 7B shows EtBr dye
displacement assay by PPI dendrimers. The inset provides an
enlargement of the graphs in the vicinity of N/P ratios which
represent the apparent ends of complexation. The highlighted areas
on the graphs demonstrate the N/P ratios, which correspond to the
apparent end of siRNA complexion by PPI G2, PPI G3, PPI G4 and PPI
G5. Means.+-.SD are shown.
[0017] FIG. 8A shows an evaluation of the siRNA complexion with PPI
dendrimers by gel retardation assay. (1) RNA size ladder; (2) naked
siRNA; (3) siRNA+PPI G2, (4) siRNA+PPI G3, (5) siRNA+PPI G4, (6)
siRNA+PPI G5. FIG. 8B provides the viability profile of A549 human
lung cancer cells incubated for 24 hours with PPI dendrimers of
different generation (from 2 to 5). Means.+-.SD are shown.
[0018] FIG. 9 provides representative AFM images of complexes
formed by siRNA in the presence of (FIG. 9A) PPI G2, (FIG. 9B) PPI
G3, (FIG. 9C) PPI G4, and (FIG. 9D) PPI G5 dendrimers after 30
minutes of complexation. The bar represents 400 nm in FIGS. 9A-9D
and 200 nm in the inset.
[0019] FIG. 10A provides representative curves which demonstrate
the size distribution of siRNA-PPI G2, siRNA-PPI G3, siRNA-PPI G4
and siRNA-PPI G5 complexes measured by DLS. Rh is the hydrodynamic
radius. FIG. 10B shows the internalization of siRNA complexed with
(1) PPI G2; (2) PPI G3; (3) PPI G4 and (4) PPI G5 dendrimers by
A549 human lung cancer cells. Intracellular fluorescence intensity
of FAM-labelled siRNA was estimated based on fluorescence
microscopy images recorded under the same experimental
conditions.
[0020] FIG. 11 provides confocal microscopy images (from left to
right: fluorescence, superimposed light and fluorescence, z-series)
of A549 human lung cancer cells incubated for 24 hours with (FIG.
11A) naked siRNA; (FIG. 11B) siRNA-PPI G2; (FIG. 11C) siRNA-PPI G3;
(FIG. 11D) siRNA-PPI G4 and (FIG. 11E) siRNA-PPI G5. Z-series
represents fluorescence images from top to the bottom of the
cell.
[0021] FIG. 12 shows the effect of incubation of A549 human lung
cancer cells with (1) medium (control); (2) siRNA-PPI G2; (3)
siRNA-PPI G3 (4) siRNA-PPI G4 and (5) siRNA-PPI G5 on the
expression of BCL2 mRNA. FIG. 12A provides a typical image of
RT-PCR products. FIG. 12B shows gene expression calculated as the
ratio of BCL2 RT-PCR product to the internal standard
(.beta..sub.2-m). Means.+-.SD are shown. *P<0.05 when compared
with control.
[0022] FIG. 13A shows the expression of BCL2 and the expression
calculated as the ratio to the internal standard (.beta..sub.2-m).
FIG. 13B provides a graph of tumor volume over time with the
various treatments: (1) Control (saline); (2) PPI dendrimer; (3)
LHRH; (4) Naked siRNA targeted to BCL2 mRNA; (5) BCL2
siRNA-PPI-DTBP-PEG; (6) LHRH-BCL2 siRNA-PPI-DTBP-PEG; (7) Free CIS;
(8) BCL2 siRNA-PPI-DTBP-PEG+CIS; (9) LHRH-BCL2
siRNA-PPI-DTBP-PEG+CIS. *P<0.05 when compared with control.
[0023] FIG. 14 provides a schematic of the synthesis of triblock
PAMAM-PEG-PLL nanocarrier.
[0024] FIG. 15 provides representative .sup.1H-NMR spectra in
D.sub.2O of PAMAM-NHAc (FIG. 15A); PAMAM-PEG-COOH (FIG. 15B);
PAMAM-PEG-PLL (FIG. 15C); and PLL-PEG-OMe (FIG. 15D).
[0025] FIG. 16 provides the characterization of different
nanocarriers and their complexes with siRNA. FIG. 16A shows the
viability of human cancer cells incubated with carriers indicated.
Means.+-.SD are shown. FIG. 16B provides representative images of
agarose gel electrophoresis of siRNA complexes with different
carriers. FIG. 16C provides average hydrodynamic diameter of
PAMAM-NHAc-PEG-PLL-siRNA complexes formed at different N/P ratio.
Means.+-.SD are shown. *P<0.05 when compared with N/P ratio
equal to 1.
[0026] FIG. 17 shows the cellular uptake and localization of naked
siRNA and PAMAM-PEG-PLL-siRNA complexes. Representative confocal
microscopy images of cancer cells incubated with fluorophore
labeled siRNA (siGLO Red, red fluorescence): naked siRNA (FIG.
17A); PAMAM-PEG-PLL-siRNA (FIG. 17B); and optical sections z-series
of cells incubated with PAMAM-PEG-PLL-siRNA (FIG. 17C).
[0027] FIG. 18 shows the serum stability of naked and complexated
siRNA. FIG. 18A provides representative images of agarose gel
electrophoreses of naked siRNA and PAMAM-PEG-PLL-siRNA complexes.
FIG. 18B provides a quantitative analysis of band intensity.
Means.+-.SD are shown.
[0028] FIG. 19 shows the expression of BCL2 gene in A2780 human
ovarian cancer cells incubated with: 1, control (fresh media); 2,
naked BCL2 siRNA; 3, naked non-specific siRNA; 4, PAMAM-BCL2 siRNA;
5, PAMAM-PEG-BCL2 siRNA; 6, PLL-BCL2 siRNA; 7, PEG-PLL-BCL2 siRNA;
8, PAMAP-PEG-PLL-BCL2 siRNA; 9, PAMAP-PEG-PLL-BCL2 non-specific
siRNA. Means.+-.SD are shown. *P<0.05 when compared with
control. .dagger.P<0.05 when compared with PLL-siRNA.
.dagger-dbl.<0.05 when compared with PAMAM-siRNA.
[0029] FIG. 20 shows the expression of CD44 mRNA (quantitative
RT-PCR) in normal and tumor tissues isolated from patients with
different gynecological cancers. Means.+-.SD are shown. P<0.05
when compared with healthy tissues from the same patient.
[0030] FIG. 21 shows suppression of CD44 mRNA (quantitative RT-PCR)
in ascitic cells obtained from patients with metastatic ovarian
cancer. Cells with incubated with the following substances:
1--Media (control); 2--Scrambled siRNA; 3--Naked CD44 siRNA;
4--CD44 siRNA delivered by non-targeted PPI dendrimer; 5--CD44
siRNA delivered by PPI dendrimer targeted to cancer cells by LHRH
peptide. Means.+-.SD are shown. P<0.05 when compared with
control.
[0031] FIG. 22 shows the suppression of CD44 protein in ascitic
cells obtained from patients with metastatic ovarian cancer. CD44
were suppressed by siRNA delivered with PPI dendrimer targeted to
cancer cells by LHRH peptide. Representative light and fluorescence
microscope images are shown.
[0032] FIG. 23 shows the cytotoxicity of different formulations.
Cells with incubated with the following substances: 1--Media
(control); 2--Scrambled siRNA; 3--Naked CD44 siRNA; 4--Free
paclitaxel; 5--CD44 siRNA delivered by non-targeted PPI dendrimer;
6--Paclitaxel delivered by non-targeted PPI dendrimer;
7--Paclitaxel and CD44 siRNA delivered by non-targeted PPI
dendrimer; 8--Paclitaxel and CD44 siRNA delivered by PPI dendrimer
targeted to cancer cells by LHRH peptide. Means.+-.SD are shown.
P<0.05 when compared with control.
[0033] FIG. 24 provides a schematic of the preparation of
tumor-targeted, stable siRNA nanoparticles. (A) Cooperative
condensation of siRNA with 5 nm SPIO nanoparticles and PPI G5
dendrimers. (B) PEGylation. (C) Conjugation of LHRH peptide to the
distal end of the PEG layer.
[0034] FIG. 25A shows siRNA complexation efficiency of SPIO
nanoparticles and PPI G5 dendrimer evaluated by the ethidium
bromide dye displacement assay. Figure inset shows enlarged
portions of the curves in the vicinity of N/P ratios which
represent the apparent ends of complexation. The circles in the
enlarged curves highlight the N/P ratios corresponding to the
apparent end of siRNA complexation by different complexation
agents. Means.+-.SD are shown. FIG. 25B provides a typical agarose
gel electrophoresis image representing siRNA complexation
efficiency by mixture of 5 nm SPIO nanoparticles and PPI G5. The
gel was stained with ethidium bromide. (1) Free siRNA (control);
(2) Mixture of 5 nm SPIO with PPI G5 and (3) Double stranded RNA
ladder.
[0035] FIG. 26A provides representative curves of the size
distribution of 5 nm SPIO-PPI G5-siRNA complexes prior and after
modification with PEG and targeting LHRH peptide measured by DLS.
Rh is hydrodynamic radius. FIG. 26B provides a representative
atomic force microscope image of the nanoparticles formed as the
result of siRNA complexation with mixture of SPIO and PPI G5. Scale
bar is equal to 400 nm.
[0036] FIG. 27 provides representative fluorescence microscopic
images of (FIG. 27A) SPIO-PPI G5-siRNA; (FIG. 27B) SPIO-PPI
G5-siRNA-PEG and (FIGS. 27C, D) SPIO-PPI G5-siRNA-PEG-LHRH
complexes after 24 hours of incubation with LHRH-positive A549
(FIGS. 27A-C) and LHRH-negative SKOV-3 (FIG. 27D) cancer cells.
siRNA was labeled with 6-FAM.
[0037] FIG. 28 provides a typical image of RT-PCR products and
expression of the BCL2 gene in human A549 (1-6) and SKOV-3 (7, 8)
cancer cells. A549 cells were treated with (1) medium (control);
(2) SPIO-PPI G5-siRNA-PEG; (3) SPIO-PPI G5-siRNA; (4) SPIO-PPI
G5-siRNA-PEG-LHRH; (5) SPIO-PPI G5; (6) SPIO-PPI G5-siRNA
(scrambled). SKOV-3 cancer cells were treated with (7) SPIO-PPI
G5-siRNA-PEG-LHRH and (8) medium (control). Gene expression was
calculated as a ratio of band intensity of studied gene to that in
internal standard (.beta.2-m, .beta.2-microglobulin). Means.+-.SD
are shown.
[0038] FIG. 29 provides representative confocal microscopy images
(light+fluorescence) of A549 human cancer cells incubated for 24
hours with SPIO-PPI G5-siRNA-PEG-LHRH (FIG. 29A) and z-series from
the top (z=10.257 .mu.m) to the bottom (z=0 .mu.m) of the single
cell (FIG. 29B).
[0039] FIG. 30 shows the cytotoxicity of (FIG. 30A) SPIO-PPI G5;
SPIO-PPI G5-siRNA; SPIO-PPI G5-siRNA-PEG-LHRH complexes and (FIG.
30B) Cisplatin; SPIO-PPI G5-siRNA+CIS; SPIO-PPI
G5-siRNA-PEG-LHRH+CIS against A549 human cancer cells. Means.+-.SD
are shown.
[0040] FIG. 31 shows the antitumor activity of different
formulations. Upper panel: typical bioluminescent images of mice
bearing subcutaneous tumor xenografts of human A549 cancer cells
transfected with luciferase. Images were taken using the IVIS
imaging system (Xenogen) in anesthetized animals at the end of the
experiment. Bottom panel: Changes of tumor volume during the
treatment. Mice were treated 3 times within 10 days with the
following formulations: (1) Control (saline); (2) LHRH; (3) siRNA;
(4) CIS; (5) SPIO-PPI-G5+CIS; (6) SPIO-PPI-G5-siRNA+CIS; (7)
LHRH-PEG-SPIO-PPI-siRNA+CIS. Means.+-.SD are shown.
DETAILED DESCRIPTION OF THE INVENTION
[0041] Drug resistance, metastases, and adverse side effects of
chemotherapy are the major causes of treatment failure in ovarian
cancer. To effectively circumvent multidrug resistance and prevent
the development of metastases in ovarian cancer, a tumor-targeted
complex delivery systems was constructed containing: (1) two
anticancer drugs with different mechanisms of action (doxorubicin
and cisplatin); (2) two antisense oligonucleotides
(ASO)--suppressors of cellular drug resistance (one ASO targeting
MDR1 and the other targeting BCL2 mRNA); and (3) a ligand
specifically targeting LHRH receptors overexpressed in the plasma
membrane of cancer cells. The tumor-targeted delivery system was
tested in a mouse xenograft model of human ovarian cancer with
intraperitoneal metastases and ascites, using tumors from patients
with ovarian cancer. The proposed treatment not only led to the
substantial regression of the growth of primary tumor, but also
prevented the development of intraperitoneal metastases and limited
adverse side effects of chemotherapy on healthy tissues.
[0042] The role of the poly(propyleneimine) (PPI) dendrimer
structure on the siRNA nanoparticles formation, facilitation of
cell internalization, and sequence specific knockdown of targeted
genes was also evaluated herein. It was found that the higher
generations of PPI dendrimers (G4 and G5) most effectively
initiated the complexation of siRNA into discrete nanoparticles
when compared with lower generations of dendrimers (G2 and G3) as
determined by tapping mode atomic force microscopy and dynamic
light scattering. The formulated siRNA-PPI dendrimer complexes
provided for a dramatic enhancement in siRNA cellular
internalization and the marked knockdown of targeted mRNA
expression in A549 human lung cancer cells. While the size and
positive charge density of G5 is much larger than G4 dendrimers,
provoking higher toxicity, G4 dendrimer shows the maximum efficacy
terms of siRNA nanoparticles formation, intracellular siRNA
internalization, and sequence specific gene silencing.
[0043] In accordance with the instant invention, methods of
treating, inhibiting, and/or preventing cancer (including, e.g.,
the inhibition or prevention of metastasis), particularly ovarian
cancer, in a patient are provided. In a particular embodiment, the
method comprises administering to the patient at least one
chemotherapeutic agent, particularly at least two chemotherapeutic
agents with different mechanisms of action, and at least one,
particularly at least two inhibitors of cellular drug resistance.
The agents may be administered within one or more liposomes and/or
dendrimers. In a particular embodiment, siRNA molecules are
delivered via dendrimers, particularly G4 or G5 PPI dendrimers. In
yet another embodiment, the at least two chemotherapeutic agents
are selected from the group consisting of doxorubicin, paclitaxel,
and cisplatin, particularly doxorubicin and cisplatin. The
inhibitors of cellular drug resistance may be pump resistance
inhibitors (drug efflux pump inhibitors) and/or nonpump resistance
inhibitors (see, e.g., Szakacs, et al. (1998) Pathol. Oncol. Res.
4:251-257; van Veen and Konings (1998) Biochem. Biophys. Acta
1365:31-36; Minko, et al. (2001) Dis. Manag. Clin. Outcomes
3:48-54; Hamaguchi, et al. (1993) Cancer Res. 53:5225-5232; Minko,
et al. (1998) J. Controlled Rel. 54:223-233; Minko, et al. (1999)
J. Controlled Rel. 59:133-148; Pakunlu, et al. (2003) Pharmaceut.
Res. 20:351-359; Alahary, et al. (1998) JPET 286:419-428; Motomura,
et al. (1998) Blood 91:3163-3171; Corrias and Tonini (1992)
Anticancer Res. 12:1431-1438; Gross, et al. (1999) Genes Dev.
13:1899-1911; Reed (1999) J. Clin. Oncol. 17:2941-2953). In a
particular embodiment, the inhibitor of pump resistance is an
inhibitor of Multi-Drug Resistance 1 (MDR1) (e.g., siRNA or
antisense) and the inhibitor of nonpump resistance is an inhibitor
of BCL2 or CD44 (e.g., siRNA or antisense). The liposomes and/or
dendrimers may further comprise at least one targeting ligand for a
cancer marker (e.g., a cell surface marker expressed only on cancer
cells or has increased expression on cancer cells compared to
normal healthy cells). In another embodiment, the targeting ligand
binds Luteinizing Hormone-Releasing Hormone (LHRH) receptor (e.g.,
an LHRH analog). Compositions comprising at least one of the above
agents and at least one pharmaceutically acceptable carrier are
also encompassed by the instant invention.
[0044] Methods of delivering a siRNA to a cell (in vitro or in
vivo) are also provided herein. In a particular embodiment, the
method comprises contacting cells with a complex comprising
siRNA(s) and a nanocarrier, particularly a dendrimer. The
nanocarrier may comprise further therapeutic agents, e.g., at least
one chemotherapeutic agent. Compositions comprising the siRNA
loaded nanocarrier (e.g., dendrimer (see, e.g., below)) and at
least one pharmaceutically acceptable carrier are also encompassed
by the instant invention. The dendrimers and compositions may be
used to treat a disease or disorder (particularly cancer) by
administering the dendrimer or composition to a patient in need
thereof, wherein the siRNA is therapeutic for said disease or
disorder.
[0045] In one embodiment, the siRNA delivery method comprises
contacting cells with a complex comprising siRNA and a cationic
dendrimer, particularly a poly(propyleneimine) (PPI) dendrimer
(e.g., a G4 or G5 dendrimer). In a particular embodiment, the
dendrimer is linked to a cancer targeting ligand (e.g., through a
PEG linker).
[0046] In another embodiment, the siRNA delivery method comprises
contacting cells with a complex comprising siRNA and a compound
comprising a polyamidoamine (PAMAM) dendrimer, polyethylene glycol
(PEG), and poly-L-lysine. In a particular embodiment, the PAMAM is
linked to the PEG which is linked the poly-L-lysine. The PAMAM may
be acetylated. In a particular embodiment, the PAMAM dendrimer is a
G4 or G5 dendrimer. In a particular embodiment, the average
molecular weight of the PEG is about 2000 to about 5000 Da,
particularly about 3000 Da. In a particular embodiment, the average
molecular weight of the poly-L-lysine is about 10,000 to about
20,000 Da, particularly about 12,000 Da. In a particular
embodiment, the compound is linked to a cancer targeting
ligand.
[0047] As stated hereinabove, the nanocarrier may comprise further
therapeutic agents, e.g., at least one chemotherapeutic agent. For
example, the nanocarriers may further comprise at least one of
doxorubicin, cisplatin, paclitaxel, topotecan, and
camptothecin.
[0048] The nanocarriers may also further comprise at least one
imaging agent. The imaging agents may be compounds useful for
optical imaging, magnetic resonance imaging (MRI), positron
emission tomography (PET), computerized tomography (CT),
gamma-scintigraphy imaging, and the like. Such agents are
well-known to those of skill in the art. Imaging agents include,
without limitation, radioisotope, isotopes, biotin and derivatives
thereof, gold (e.g., nanoparticles), optical imaging agents (e.g.,
near IR dyes (e.g., IRDye 800CW) phorphyrins, anthraquinones,
anthrapyrazoles, perylenequinones, xanthenes, cyanines, acridines,
phenoxazines, phenothiazines and derivatives thereof), chromophore,
fluorescent compounds (e.g., Alexa Fluor.RTM. 488, fluorescein,
rhodamine, DiI, DiO, and derivatives thereif), MRI enhancing agents
(for example, DOTA-Gd3.sup.+
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra (acetic acid)),
DTPA-Gd3.sup.+ (gadolinium complex with diethylenetriamine
pentaacetic acid), etc.), paramagnetic or superparamagnetic ions
(e.g., Gd(III), Eu(III), Dy(III), Pr(III), Pa(IV), Mn(II), Cr(III),
Co(III), Fe(III), Cu(II), Ni(II), Ti(III), and V(IV)), PET
compounds labeled or complexed with .sup.11C, .sup.13N, .sup.15O,
.sup.18F, .sup.64Cu, .sup.68Ga, or .sup.82Rb (e.g., .sup.18F-FDG
(fluorodeoxyglucose)), CT compounds (for example, iodine or barium
containing compounds, e.g., 2,3,5-triiodobenzoic acid), and gamma
or positron emitters (for example, .sup.99mTc, .sup.111In,
.sup.113In, .sup.153Sm, .sup.123I, .sup.131I .sup.18F, .sup.64Cu,
.sup.201Tl, etc.). In a particular embodiment, the imaging agent is
an MRI contrast agent, particularly superparamagnetic iron oxide
(SPIO).
[0049] A targeting ligand is a compound that will specifically bind
to a specific type of tissue or cell type. The term "cancer marker"
refers to biomolecules (e.g., proteins, carbohydrates,
glycoproteins, and the like) that are exclusively or preferentially
or differentially expressed on a cancer cell and thereby provide
targets preferential or specific to the cancer. The preferential
expression can be preferential expression as compared to any other
cell in the organism or preferential expression within a particular
area/organ/tissue of the organism. As used herein, the term "cancer
targeting ligand" refers to a targeting ligand that specifically
binds a cancer marker. A "cancer targeting ligand" may be an
antibody immunologically specific for the cancer marker. For
example, Her2 is a well known breast cancer marker. Trastuzumab is
a monoclonal antibody which specifically binds the extracellular
domain of HER2 and is a cancer targeting ligand. Epidermal growth
factor receptor (EGFR) is also a well known cancer marker. In a
particular embodiment of the instant invention, the cancer
targeting ligand is specific for LHRH receptors (LHRHR). In a
particular embodiment, the cancer targeting ligand is LHRH or an
analog thereof (e.g., SEQ ID NO: 3). In a particular embodiment,
the cancer targeting ligand is a CD44 antibody or fragment
thereof.
[0050] Cancers that may be treated using the present invention
(e.g., siRNA delivery vehicles) include, but are not limited to:
cancers of the prostate, colorectum, pancreas, cervix, stomach,
endometrium, brain, liver, bladder, ovary, testis, head, neck, skin
(including melanoma and basal carcinoma), mesothelial lining, white
blood cell (including lymphoma and leukemia) esophagus, breast,
muscle, connective tissue, lung (including small-cell lung
carcinoma and non-small-cell carcinoma), adrenal gland, thyroid,
kidney, or bone; glioblastoma, mesothelioma, renal cell carcinoma,
gastric carcinoma, sarcoma, choriocarcinoma, cutaneous basocellular
carcinoma, skin squamous cell carcinomas, and testicular seminoma.
In a particular embodiment, the cancer is a gynecological cancer
(e.g., cervical, ovarian, uterine, vaginal, and vulvar). In a
particular embodiment, the cancer is ovarian cancer. In a
particular embodiment, the compositions of the instant invention
are co-administered with chemotherapy (e.g., radiation). The
compositions of the instant invention may be administered before,
after, and/or simultaneously with the other agents or therapy.
I. DEFINITIONS
[0051] The following definitions are provided to facilitate an
understanding of the present invention:
[0052] The term "isolated" may refer to protein, nucleic acid,
compound, or cell that has been sufficiently separated from the
environment with which it would naturally be associated, so as to
exist in "substantially pure" form. "Isolated" does not necessarily
mean the exclusion of artificial or synthetic mixtures with other
compounds or materials, or the presence of impurities that do not
interfere with the fundamental activity, and that may be present,
for example, due to incomplete purification.
[0053] "Pharmaceutically acceptable" indicates approval by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans.
[0054] A "carrier" refers to, for example, a diluent, adjuvant,
preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g.,
ascorbic acid, sodium metabisulfite), solubilizer (e.g., Tween 80,
Polysorbate 80), emulsifier, buffer (e.g., Tris HCl, acetate,
phosphate), bulking substance (e.g., lactose, mannitol), excipient,
auxilliary agent, filler, disintegrant, lubricating agent, binder,
stabilizer, preservative or vehicle with which an active agent of
the present invention is administered. Pharmaceutically acceptable
carriers can be sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil and the like.
Water or aqueous saline solutions and aqueous dextrose and glycerol
solutions are preferably employed as carriers, particularly for
injectable solutions. The compositions can be incorporated into
particulate preparations of polymeric compounds such as polylactic
acid, polyglycolic acid, etc., or into liposomes or micelles. Such
compositions may influence the physical state, stability, rate of
in vivo release, and rate of in vivo clearance of components of a
pharmaceutical composition of the present invention. The
pharmaceutical composition of the present invention can be
prepared, for example, in liquid form, or can be in dried powder
form (e.g., lyophilized). Suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin
(Mack Publishing Co., Easton, Pa.); Gennaro, A. R., Remington: The
Science and Practice of Pharmacy, 20th Edition, (Lippincott,
Williams and Wilkins), 2000; Liberman, et al., Eds., Pharmaceutical
Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe, et
al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.),
American Pharmaceutical Association, Washington, 1999.
[0055] "Nucleic acid" or a "nucleic acid molecule" as used herein
refers to any DNA or RNA molecule, either single or double stranded
and, if single stranded, the molecule of its complementary sequence
in either linear or circular form. In discussing nucleic acid
molecules, a sequence or structure of a particular nucleic acid
molecule may be described herein according to the normal convention
of providing the sequence in the 5' to 3' direction. With reference
to nucleic acids of the invention, the term "isolated nucleic acid"
is sometimes used. This term, when applied to DNA, refers to a DNA
molecule that is separated from sequences with which it is
immediately contiguous in the naturally occurring genome of the
organism in which it originated. For example, an "isolated nucleic
acid" may comprise a DNA molecule inserted into a vector, such as a
plasmid or virus vector, or integrated into the genomic DNA of a
prokaryotic or eukaryotic cell or host organism.
[0056] When applied to RNA, the term "isolated nucleic acid" refers
primarily to an RNA molecule encoded by an isolated DNA molecule as
defined above. Alternatively, the term may refer to an RNA molecule
that has been sufficiently separated from other nucleic acids with
which it would be associated in its natural state (i.e., in cells
or tissues). An "isolated nucleic acid" (either DNA or RNA) may
further represent a molecule produced directly by biological or
synthetic means and separated from other components present during
its production.
[0057] A "replicon" is any genetic element, for example, a plasmid,
cosmid, bacmid, plastid, phage or virus, which is capable of
replication largely under its own control. A replicon may be either
RNA or DNA and may be single or double stranded.
[0058] A "vector" is a replicon, such as a plasmid, cosmid, bacmid,
phage or virus, to which another genetic sequence or element
(either DNA or RNA) may be attached so as to bring about the
replication of the attached sequence or element.
[0059] The terms "percent similarity", "percent identity" and
"percent homology" when referring to a particular sequence are used
as set forth in the University of Wisconsin GCG software
program.
[0060] The term "substantially pure" refers to a preparation
comprising at least 50-60% by weight of a given material (e.g.,
nucleic acid, oligonucleotide, protein, etc.). More preferably, the
preparation comprises at least 75% by weight, and most preferably
90-95% by weight of the given compound. Purity is measured by
methods appropriate for the given compound (e.g. chromatographic
methods, agarose or polyacrylamide gel electrophoresis, HPLC
analysis, and the like).
[0061] The term "oligonucleotide" as used herein refers to
sequences, primers and probes of the present invention, and is
defined as a nucleic acid molecule comprised of two or more ribo-
or deoxyribonucleotides, preferably more than three. The exact size
of the oligonucleotide will depend on various factors and on the
particular application and use of the oligonucleotide.
[0062] Antisense molecules are oligonucleotides that hybridize
under physiological conditions to a particular gene or to an mRNA
transcript of such gene and, thereby, inhibit the transcription of
such gene and/or the translation of such mRNA. The antisense
molecules are designed so as to interfere with transcription or
translation of a target gene upon hybridization with the target
gene or its mRNA. Antisense molecules are typically between about
15 and about 30 nucleotides, but the exact length of the antisense
oligonucleotide and its degree of complementarity with its target
depend upon the specific target selected. An antisense
oligonucleotide is preferably constructed to bind selectively with
the target nucleic acid under physiological conditions. Antisense
molecules may span the translational start site of mRNA molecules.
Antisense constructs may also be generated which contain the entire
gene sequence in reverse orientation. Antisense oligonucleotides
targeted to any known nucleotide sequence can be prepared by
oligonucleotide synthesis according to standard methods.
[0063] The term "siRNA" refers to small inhibitory RNA duplexes
such as those that induce the RNA interference (RNAi) pathway.
siRNA may vary in length, but are generally 12-30, more typically
about 21 nucleotides in length (see Ausubel et al., eds., Current
Protocols in Molecular Biology, John Wiley and Sons, Inc.). siRNA
may have unpaired overhanging bases on the 5' or 3' end of the
sense strand and/or the antisense strand. As used herein, the term
"siRNA" includes duplexes of two separate strands and single strand
molecules that can form hairpin structures comprising a duplex
region (shRNA).
[0064] With respect to single stranded nucleic acids, particularly
oligonucleotides, the term "specifically hybridizing" refers to the
association between two single-stranded nucleotide molecules of
sufficiently complementary sequence to permit such hybridization
under pre-determined conditions generally used in the art
(sometimes termed "substantially complementary"). In particular,
the term refers to hybridization of an oligonucleotide with a
substantially complementary sequence contained within a
single-stranded DNA molecule of the invention, to the substantial
exclusion of hybridization of the oligonucleotide with
single-stranded nucleic acids of non-complementary sequence.
Appropriate conditions enabling specific hybridization of single
stranded nucleic acid molecules of varying complementarity are well
known in the art.
[0065] For instance, one common formula for calculating the
stringency conditions required to achieve hybridization between
nucleic acid molecules of a specified sequence homology is set
forth below (Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press):
T.sub.m=81.5.degree. C.+16.6 Log [Na+]+0.41(% G+C)-0.63 (%
formamide)-600/#bp in duplex
[0066] As an illustration of the above formula, using [Na+]=[0.368]
and 50% formamide, with GC content of 42% and an average probe size
of 200 bases, the T.sub.m is 57.degree. C. The T.sub.m of a DNA
duplex decreases by 1-1.5.degree. C. with every 1% decrease in
homology. Thus, targets with greater than about 75% sequence
identity would be observed using a hybridization temperature of
42.degree. C.
[0067] The stringency of the hybridization and wash depend
primarily on the salt concentration and temperature of the
solutions. In general, to maximize the rate of annealing of the
probe with its target, the hybridization is usually carried out at
salt and temperature conditions that are 20-25.degree. C. below the
calculated T.sub.m of the hybrid. Wash conditions should be as
stringent as possible for the degree of identity of the probe for
the target. In general, wash conditions are selected to be
approximately 12-20.degree. C. below the T.sub.m of the hybrid. In
regards to the nucleic acids of the current invention, a moderate
stringency hybridization is defined as hybridization in
6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/ml
denatured salmon sperm DNA at 42.degree. C., and washed in
2.times.SSC and 0.5% SDS at 55.degree. C. for 15 minutes. A high
stringency hybridization is defined as hybridization in
6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/ml
denatured salmon sperm DNA at 42.degree. C., and washed in
1.times.SSC and 0.5% SDS at 65.degree. C. for 15 minutes. A very
high stringency hybridization is defined as hybridization in
6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/ml
denatured salmon sperm DNA at 42.degree. C., and washed in
0.1.times.SSC and 0.5% SDS at 65.degree. C. for 15 minutes.
[0068] Chemotherapeutic agents are compounds that exhibit
anticancer activity and/or are detrimental to a cell (e.g., a
toxin). Suitable chemotherapeutic agents include, but are not
limited to (along with mechanism of action): toxins (e.g., saporin,
ricin, abrin, ethidium bromide, diptheria toxin, Pseudomonas
exotoxin); taxanes; alkylating agents (e.g., nitrogen mustards such
as chlorambucil, cyclophosphamide, isofamide, mechlorethamine,
melphalan, and uracil mustard; aziridines such as thiotepa;
methanesulphonate esters such as busulfan; nitroso ureas such as
carmustine, lomustine, and streptozocin; platinum complexes (e.g.,
cisplatin, carboplatin, tetraplatin, ormaplatin, thioplatin,
satraplatin, nedaplatin, oxaliplatin, heptaplatin, iproplatin,
transplatin, and lobaplatin); bioreductive alkylators (e.g.,
mitomycin, procarbazine, dacarbazine and altretamine); DNA
strand-breakage agents (e.g., bleomycin); topoisomerase II
inhibitors (e.g., amsacrine, menogaril, amonafide, dactinomycin,
daunorubicin, N,N-dibenzyl daunomycin, ellipticine, daunomycin,
pyrazoloacridine, idarubicin, mitoxantrone, m-ANSA, doxorubicin,
deoxyrubicin, etoposide, etopside phosphate, oxanthrazole,
rubidazone, epirubicin, bleomycin, and teniposide); DNA minor
groove binding agents (e.g., plicamydin); antimetabolites (e.g.,
folate antagonists such as methotrexate and trimetrexate);
pyrimidine antagonists such as fluorouracil, fluorodeoxyuridine,
CB3717, azacitidine, cytarabine, and floxuridine; purine
antagonists such as mercaptopurine, 6-thioguanine, fludarabine,
pentostatin; asparginase; and ribonucleotide reductase inhibitors
such as hydroxyurea); tubulin interactive agents (e.g.,
vincristine, vinblastine, and paclitaxel (Taxol)); hormonal agents
(e.g., estrogens; conjugated estrogens; ethinyl estradiol;
diethylstilbesterol; chlortrianisen; idenestrol; progestins such as
hydroxyprogesterone caproate, medroxyprogesterone, and megestrol;
and androgens such as testosterone, testosterone propionate,
fluoxymesterone, and methyltestosterone); adrenal corticosteroids
(e.g., prednisone, dexamethasone, methylprednisolone, and
prednisolone); leutinizing hormone releasing agents or
gonadotropin-releasing hormone antagonists (e.g., leuprolide
acetate and goserelin acetate); and antihormonal antigens (e.g.,
tamoxifen, antiandrogen agents such as flutamide; and antiadrenal
agents such as mitotane and aminoglutethimide).
II. ADMINISTRATION
[0069] The compositions described herein will generally be
administered to a patient as a pharmaceutical preparation. The term
"patient" as used herein refers to human or animal subjects. These
compositions may be employed therapeutically, under the guidance of
a physician.
[0070] The compositions of the instant invention may be
conveniently formulated for administration with any
pharmaceutically acceptable carrier(s). For example, the agents may
be formulated with an acceptable medium such as water, buffered
saline, ethanol, polyol (for example, glycerol, propylene glycol,
liquid polyethylene glycol and the like), dimethyl sulfoxide
(DMSO), oils, detergents, suspending agents or suitable mixtures
thereof. The concentration of the agents in the chosen medium may
be varied and the medium may be chosen based on the desired route
of administration of the pharmaceutical preparation. Except insofar
as any conventional media or agent is incompatible with the agents
to be administered, its use in the pharmaceutical preparation is
contemplated.
[0071] The dose and dosage regimen of compositions according to the
invention that are suitable for administration to a particular
patient may be determined by a physician considering the patient's
age, sex, weight, general medical condition, and the specific
condition for which the composition is being administered and the
severity thereof. The physician may also take into account the
route of administration, the pharmaceutical carrier, and the
composition's biological activity.
[0072] Selection of a suitable pharmaceutical preparation will also
depend upon the mode of administration chosen. For example, the
compositions of the invention may be administered by direct
injection to a desired site. In this instance, a pharmaceutical
preparation comprises the agents dispersed in a medium that is
compatible with the site of injection.
[0073] Compositions of the instant invention may be administered by
any method. For example, the compositions of the instant invention
can be administered, without limitation parenterally,
subcutaneously, orally, topically, pulmonarily, rectally,
vaginally, intravenously, intraperitoneally, intrathecally,
intracerbrally, epidurally, intramuscularly, intradermally, or
intracarotidly. In a particular embodiment, the compositions are
administered intravenously or intraperitoneally. Pharmaceutical
preparations for injection are known in the art. If injection is
selected as a method for administering the composition, steps must
be taken to ensure that sufficient amounts of the molecules reach
their target cells to exert a biological effect.
[0074] Pharmaceutical compositions containing an agent of the
present invention as the active ingredient in intimate admixture
with a pharmaceutically acceptable carrier can be prepared
according to conventional pharmaceutical compounding techniques.
The carrier may take a wide variety of forms depending on the form
of preparation desired for administration, e.g., intravenous, oral,
direct injection, intracranial, and intravitreal.
[0075] A pharmaceutical preparation of the invention may be
formulated in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form, as used herein, refers to a
physically discrete unit of the pharmaceutical preparation
appropriate for the patient undergoing treatment. Each dosage
should contain a quantity of active ingredient calculated to
produce the desired effect in association with the selected
pharmaceutical carrier. Procedures for determining the appropriate
dosage unit are well known to those skilled in the art.
[0076] Dosage units may be proportionately increased or decreased
based on the weight of the patient. Appropriate concentrations for
alleviation of a particular pathological condition may be
determined by dosage concentration curve calculations, as known in
the art.
[0077] In accordance with the present invention, the appropriate
dosage unit for the administration of compositions of the instant
invention may be determined by evaluating the toxicity of the
molecules or cells in animal models. Various concentrations of
agents in pharmaceutical preparations may be administered to mice,
and the minimal and maximal dosages may be determined based on the
beneficial results and side effects observed as a result of the
treatment. Appropriate dosage unit may also be determined by
assessing the efficacy of the agent treatment in combination with
other standard drugs. The dosage units of the compositions may be
determined individually or in combination with each treatment
according to the effect detected.
[0078] The pharmaceutical preparation comprising the agents of the
instant invention may be administered at appropriate intervals, for
example, at least twice a day or more until the pathological
symptoms are reduced or alleviated, after which the dosage may be
reduced to a maintenance level. The appropriate interval in a
particular case would normally depend on the condition of the
patient.
[0079] The instant invention encompasses methods of treating a
disease/disorder comprising administering to a subject in need
thereof a composition comprising an agent of the instant invention
and, preferably, at least one pharmaceutically acceptable carrier.
In a particular embodiment, the disease is cancer, particularly
ovarian cancer. Other methods of treating the disease or disorder
may be combined with the methods of the instant invention (e.g.,
other chemotherapeutic agents or therapy (e.g., radiation)) may be
co-administered with the compositions of the instant invention.
[0080] The following examples provide illustrative methods of
practicing the instant invention, and are not intended to limit the
scope of the invention in any way.
Example 1
[0081] A tumor-targeted liposomal drug delivery system (DDS) was
constructed containing doxorubicin (DOX) or cisplatin (CIS) as
anticancer drugs, a synthetic analog of Luteinizing
Hormone-Releasing Hormone (LHRH) as tumor targeting moiety, and
Antisense Oligonucleotides (ASO) targeted to MDR1 and BCL2 mRNA as
suppressors of pump and nonpump resistance, respectively. The DDS
was tested in a mouse xenograft model of human metastatic ovarian
cancer with intraperitoneal metastases. Primary tumors were
developed by subcutaneous injection of cancer cells isolated from
fresh malignant ascites of patients with advanced ovarian
carcinoma.
Materials and Methods
Materials
[0082] Egg phosphatidylcholin (EPC), Cholesterol (Chol), and
1,2,-distearoyl-sn-glycero-3-phosphoethanolamine-N-aminopolyethelenglycol-
--Mw--2000 ammonium salt (DSPE-PEG) were purchased from Avanti
Polar Lipids (Alabaster, Ala.); P-ethoxy modified antisense
oligonucleotides targeted to MDR1 (5'-TTC AAG ATC CAT CCC GAC CTC
GCG-3'; SEQ ID NO: 1) and BCL2 (5'-CAG CGT GCG CCA TCC TTC CC-3';
SEQ ID NO: 2) mRNA were synthesized as described (Pakunlu, et al.
(2003) Pharm. Res., 20:351-359; Pakunlu et al. (2004) Cancer Res.,
64:6214-6224; Pakunlu et al. (2006) J. Control. Rel., 114:153-162)
by Oligos Etc. (Wilson, Oreg.); cisplatin and doxorubicin were
purchased from Sigma (St. Louis, Mo.). A synthetic analog of LHRH,
Lys6-des-Gly10-Pro9-ethylamide
(Gln-His-Trp-Ser-Tyr-DLys-Leu-Arg-Pro-NH-Et; SEQ ID NO: 3) was
synthesized as described (Chandna et al. (2007) Mol. Pharm.,
4:668-678; Dharap et al. (2005) Proc. Natl. Acad. Sci.,
102:12962-12967; Pakunlu et al. (2006) J. Control. Rel.,
114:153-162; Dharap et al. (2003) J. Control. Rel., 91:61-73;
Garbuzenko et al. (2009) Pharm. Res., 26:382-394; Khandare et al.
(2006) J. Pharmacol. Exp. Ther., 317:929-937; Saad et al. (2008) J.
Control. Rel., 130:107-114) by American Peptide (Sunnyvale,
Calif.). For in vitro experiments, LHRH peptide was labeled by
Rhodamine (Invitrogen Molecular Probs, Carlsbad, Calif.) as
previously described (Khandare et al. (2006) J. Pharmacol. Exp.
Ther., 317:929-937).
Drug Formulations
[0083] PEGylated "neutral" liposomes were prepared as previously
described using lipids (EPC, Cholesterol, and DSPE-PEG in ratio
51:44:5, respectively) (Pakunlu et al. (2006) J. Control Release
114:153-162; Garbuzenko et al. (2009) Pharm. Res., 26:382-394). To
prepare tumor-targeted liposomes, DSPE-PEG was conjugated with LHRH
peptide as previously described (Saad et al. (2008) J. Control.
Release 130:107-114) and added together with regular DSPE-PEG. To
prepare CIS loaded liposomes, thin film layer was rehydrated in
0.9% NaCl containing 2 mg/ml of CIS. It is known that both siRNA
and ASO can be effectively used for the suppression of targeted
genes and proteins (Betigeri et al. (2006) Mol. Pharm. 3:424-430).
"Neutral" liposomes cannot be used for the effective delivery of
negatively charged siRNA. The delivery of siRNA requires cationic
liposomes. However, cationic liposomes are not the best choice for
the delivery of DOX. To use "neutral" liposomes as a delivery
vehicle for drugs and nucleic acids, a modified "neutral" form of
ASO was used. Unfortunately, it is not possible to neutralize the
charge of siRNA without substantial decrease in their activity. ASO
targeted to BCL2 and MDR1 mRNA were dissolved in 0.9% NaCl in
concentrations at 0.25 mM of each ASO (Pakunlu et al. (2006) J.
Control Release 114:153-162). To prepare DOX loaded liposomes, the
thin film layer was rehydrated with 300 mM citrate buffer (pH=4)
followed by overnight dialysis against 0.9% NaCl at 4.degree. C.,
and incubated with DOX solution (5 mg/ml) at 37.degree. C. for 40
minutes (Garbuzenko et al. (2009) Pharm. Res., 26:382-394). In most
cases, DOX-loaded liposomes preparation is based on transmembrane
ammonium ion gradient. The other way to create transmembrane
gradient includes the use of two buffers with different pH for
internal and external liposomal areas. Citric acid buffer, 300 mM
with the pH and 0.9% (150 mM) NaCl with pH .about.5.5, was used,
respectively (Wasserman et al. (2007) Langmuir 23:1937-1947). The
choice was based on the following consideration. Liposomes were
passively loaded with ASO which should have been dissolved in the
rehydration buffer and mixed with the lipids. The preliminary data
showed that citric acid buffer with pH 4 was more effective and
safe for loading of ASO when compared with NH.sub.4CL solution.
Before loading, DOX was dissolved in double distilled water.
Liposomes were separated from free drugs and ASO by overnight
dialysis against 0.9% NaCl. The aliquots of liposomes were
destroyed in isopropanol in a ratio of 10:90
(liposomes:isopropanol) and the concentration of trapped DOX was
determined by high-performance liquid chromatography using a
symmetry C18 column (150 mm.times.4.6 mm, Waters Corporation,
Milford, Mass., USA) operated at room temperature. The mobile phase
consisted of 0.1% trifluoroacetic acid in water/acetonitrile 25:75
v:v; the flow rate was set to 1.0 ml/min, wavelength 480 nm. The
chromatographic instillation consisted of a Model 1525 pump (Waters
Corporation, Milford, Mass., USA), a Model 717 Plus auto-injector
(Waters Corporation) and a Model 2487 variable wavelength UV
detector (Waters Corporation) connected to the Millennium software.
Loading efficacy of DOX was about 90% from total amount. The size
of liposomes was measured by dynamic light scattering using 90Plus
Particle Size Analyzer (Brookhaven Instruments, New York, N.Y.).
The average size of EPC/Cholesterol/DSPE-PEG liposomes loaded with
drugs and ASO was 130.+-.10 nm. Electrical charge of liposomes was
measured using ZetaPALS Zeta Potential Analyzer (Brookhaven
Instruments, New York, N.Y.). The average zeta potential of empty
and loaded PEGylated liposomes was -10.+-.2 mV.
Cancer Cells
[0084] Discarded de-identified pathological materials obtained from
the Cancer Institute of New Jersey were used to isolate cancer
cells from tissues obtained from patients with ovarian carcinoma.
The ascites fluid with cancer cells was obtained from the
peritoneal area of patients with ovarian cancer. The samples were
centrifuged for 20 minutes at 2000 g; the supernatant was discarded
and cell pellets were consequently resuspended. The resuspended
cells were cultured in RPMI media (Sigma, St. Louis, Mo.)
supplemented with fetal bovine serum (Fisher Chemicals, Fairlawn,
N.J.), 2.5 .mu.g/ml insulin and 1.2 ml/100 ml
penicillin-streptomycin (Sigma, St. Louis, Mo.).
Generation of Ascites Cells Stably Expressing Luciferase
[0085] In order to visualize cells and document tumor growth,
ascitic cells were transfected with luciferase or green fluorescent
protein. Briefly, cells were transfected for 24 hours in a 6-well
plate with 2.0 .mu.g of pMetLuc-Control vector (Clontech, Mountain
View, Calif.) containing the neomycin resistance gene using
Lipofectamine.TM. 2000 (Invitrogen, Carlsbad, Calif.) for
luciferase or pAcGFP1-C1 vector (Clontech, Mountain View, Calif.)
for green fluorescent protein according to the manufacture's
recommendations. Cells were maintained in media containing 500
.mu.g/ml G418 (Gibco, Grand Island, N.Y.) for further study. Cells
were grown at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2 (v/v) in air. All experiments were performed on cells in
the exponential growth phase.
Animal Model and In Vivo Antitumor Activity
[0086] An animal model of human ovarian carcinoma xenografts was
created as previously described (Chandna et al. (2007) Mol. Pharm.,
4:668-678; Dharap et al. (2005) Proc. Natl. Acad. Sci.,
102:12962-12967; Chandna et al. (2010) Pharm. Res.; Pakunlu et al.
(2006) J. Control. Rel., 114:153-162; Saad et al. (2008) J.
Control. Rel., 130, 107-114; Dharap et al. (2006) J. Pharmacol.
Exp. Ther., 316:992-998; Minko et al. (2000) Int. J. Cancer,
86:108-117; Minko et al. (2000) Pharm. Res., 17:505-514; Wang et
al. (2008) Clin. Cancer Res., 14:3607-3616). Briefly, human ascitic
cells (2.times.10.sup.6) were subcutaneously transplanted into the
flanks of female athymic nu/nu mice (NCRNU-M,
CrTac:NCr-Foxn1.sup.nu, Taconic Farms, Inc, Cranbury, N.J.). All
mice received cancer cells from the same patient. When the tumors
reached a size of about 0.3 cm.sup.3 (15-20 days after
transplantation), mice were treated intraperitoneally with the
following formulations: (1) Saline (untreated control); (2)
Liposomes (Lip); (3) LHRH; (4) DOX; (5) Lip-DOX; (6) Lip-DOX-BCL2
ASO; (7) Lip-DOX-MDR1 ASO; (8) Lip-DOX-BCL2-MDR1 ASO; (9)
LHRH-Lip-DOX-BCL2-MDR1 ASO; (10) CIS; (11) Lip-CIS; (12)
Lip-CIS-BCL2 ASO; (13) Lip-CIS-MDR1 ASO; (14) Lip-CIS-BCL2-MDR1
ASO; (15) LHRH-Lip-CIS-BCL2-MDR1 ASO; (16) DOX+CIS; (17)
Lip-DOX+Lip-CIS; (18) Lip-DOX-BCL2 ASO+Lip-CIS-BCL2 ASO; (19)
Lip-DOX-MDR1 ASO+Lip-CIS-MDR1 ASO; (20) Lip-DOX-BCL2-MDR1
ASO+Lip-CIS-BCL2-MDR1 ASO; (21) LHRH-Lip-DOX-BCL2-MDR1
ASO+LHRH-Lip-CIS-BCL2-MDR1 ASO. The doses of DOX (2.5 mg/kg) and
CIS (2.5 mg/kg) in formulations corresponded to the maximum
tolerated dose of these drugs. The maximum tolerated doses were
estimated in separate experiments based on the animal weight change
after the injection of increasing doses of drugs as previously
described (Chandna et al. (2007) Mol. Pharm., 4:668-678; Dharap et
al. (2005) Proc. Natl. Acad. Sci., 102:12962-12967; Khandare et al.
(2006) J. Pharmacol. Exp. Ther., 317:929-937; Dharap et al. (2006)
J. Pharmacol. Exp. Ther., 316:992-998). The animals were treated
maximum 8 times over four weeks and the development of primary
tumor and intraperitoneal metastases was monitored by fluorescent
IVIS (Xenogen, Alameda, Calif.) and ultrasound Vevo 2100
(VisualSonics, Toronto, Canada) imaging systems. To initiate
bioluminescence of cancer cells, 1.times. substrate/reaction buffer
(Clontech, Mountain View, Calif.) was diluted 1:20 with DPBS and
100 .mu.l of the solution was injected into each mouse. The
bioluminescent images were taken 10 minutes after the injection of
the substrate. The size of primary tumor was measured by a caliper.
At the end of the experiments, tumors and ascites were excised and
their mass was measured. Animal weight was evaluated every day
during the treatment period. Changes in tumor size were used as an
overall marker for antitumor activity. According to the protocol
approved by the Rutgers University Animal Care and Facilities
Committee, animals were euthanized when tumor volume reached
approximately 2,000 mm.sup.3 (about 10% of body weight) or when
body weight significantly changed when compared with the control
untreated group.
Expression of Targeted Genes and Proteins
[0087] The expression of MDR1 and BCL2 genes was measured using a
quantitative RT-PCR as previously described (Pakunlu et al. (2006)
J. Control. Rel., 114:153-162). Gene expression was calculated as a
percent of internal standard (.beta..sub.2-microglobulin). To
identify the presence of BCL2, P-glycoprotein and Caspase 3 (CASP3)
proteins, the immunohistochemical staining of paraffin-embedded
tumor tissue slides was carried out. For BCL2 and CASP3, slides
were deparaffinized in xylene for 5 min followed by progressive
rehydration in 100%, 95%, 70% and 50% ethanol for 3 minutes during
each step. Endogenous peroxidase activity was blocked by incubating
slides in 3% H.sub.2O.sub.2 solution in methanol at room
temperature for 10 minutes and washing in 300 ml PBS two times for
5 minutes. Slides then were stained with anti-mouse monoclonal
antibodies for BCL2 conjugated with FITC (1:200 dilution;
BioLegend, catalog number 633502, San Diego, Calif.) and for CASP3
conjugated with Alexa Flour 647 (1:200 dilution; BioLegend, catalog
number 622702, San Diego, Calif.) by incubating for an hour, washed
in 300 ml PBS two times for 5 minutes and analyzed by fluorescence
microscopy. To identify the expression of P-glycoprotein, after
deparaffinization and rehydration, the slides were stained using
Vector.RTM. M.O.M. Immunodetection Kit (Vector Laboratories, Inc.,
Burlingame, Calif.). Mouse monoclonal antibody to P-glycoprotein
(ab3366, 1:40 dilution) obtained from Abcam (Cambridge, Mass.) was
used as primary antibody for the detection of P-glycoprotein.
Biotinylated anti-mouse IgG Reagent (1:250 dilution, Vector
Laboratories, Inc., Burlingame, Calif.) and HSP-Streptavidine
Detection System (1:500 dilution, Vector Laboratories, Inc.,
Burlingame, Calif.) in combination with DAB substrate kit (Vector
Laboratories, Inc., Burlingame, Calif.) for peroxidase were used
for visualization. After staining, the slides were examined by
light microscopy and photographed.
Histopathologic Analysis
[0088] After sacrificing an animal, the tumors and organs were
extracted and immediately fixed in 10% phosphate-buffered formalin.
Samples were subsequently dehydrated and embedded in
Paraplast.RTM.. Five-micrometer sections were cut and stained with
hematoxylin-eosin as previously described (Lu et al. (2006) Cancer
Res., 66:11494-11501) and analyzed.
Apoptosis
[0089] Induction of apoptosis was analyzed by the measurement of
the enrichment of histone-associated DNA fragments (mono- and
oligo-nucleosomes) in homogenates of the tumor, malignant ascites
and other organs (liver, kidney, lung, heart and brain) using
anti-histone and anti-DNA antibodies by a cell death detection
ELISA Plus kit (Roche, Nutley, N.J.) as previously described
(Pakunlu et al. (2004) Cancer Res., 64:6214-6224; Dharap et al.
(2005) Proc. Natl. Acad. Sci., 102:12962-12967; Dharap et al.
(2003) Pharm. Res., 20:889-896).
Statistical Analysis
[0090] Data obtained were analyzed using descriptive statistics,
single factor analysis of variance (ANOVA) and presented as a mean
value.+-.standard deviation (SD) from ten independent measurements.
Data sets were analyzed for significance with Student's t test and
considered P value of less than 0.05 as statistical
significant.
Results
Development of Primary Solid Tumors and Intraperitoneal
Metastases
[0091] To create a mouse model of human ovarian carcinoma, cells
isolated from human malignant ascites obtained from patients with
advanced ovarian cancer were transfected with luciferase or green
fluorescent protein (FIG. 1A) and subcutaneously injected in the
flank of nude mice (FIG. 1B). Transfection of cancer cells with
luciferase allowed for their visualization in live anesthetized
animals using a bioluminescence IVIS imaging system. In untreated
animals, the growth of primary solid subcutaneous tumors in 80% of
animals was accompanied by the development of malignant
intraperitoneal ascites and carcinomatosis (FIG. 1C). In addition
to bioluminescence visualization, the existence of intraperitoneal
metastases and malignant ascites was also confirmed in live animals
using ultrasound Vevo imaging system (FIGS. 1D and 1F).
[0092] To deliver anticancer drugs specifically to tumor cells, the
LHRH peptide was used as a targeting moiety. LHRH receptors (LHRHR)
are overexpressed in many types of cancer cells and are not
expressed in a detectable level in visceral organs (FIG. 1H).
Although the expression of these receptors was found in tissues of
healthy non-tumorous reproductive organs, the expression of LHRHR
in tumor cells was significantly higher when compared with
corresponding healthy tissue taken from the same organ of the same
patient (FIG. 1I). To determine whether LHRHR are overexpressed in
cells isolated from human malignant ascites, ascitic cells were
incubated with LHRH peptide labeled with Rhodamine and registered
its fluorescence by fluorescence microscopy. The fluorescence of
labeled LHRH peptide was observed predominantly in the plasma
membrane of cancer cells (FIG. 1E) where LHRH receptors are
localized. In addition, the expression of mRNA encoded LHRHR was
measured in cells isolated from human malignant ascites and found
that these receptors were overexpressed in malignant ascites (FIG.
1G). Consequently, LHRH peptide can be used for targeting cancer
cells in both primary tumors and intraperitoneal metastases. A
complex tumor-targeted proapoptotic drug delivery system was
constructed that contained PEGylated liposomes as carriers, LHRH
peptides conjugated to distal ends of PEG polymers as targeting
moieties, CIS or DOX as anticancer drugs-cell death inducers, and
MDR1 and BCL2 antisense oligonucleotides (ASO) as suppressors of
pump and nonpump cellular resistance, respectively (FIG. 1J). CIS,
as a substance with low aqueous solubility, was located in the
phospholipid bilayer of the liposomal membrane, while water-soluble
DOX and P-ethoxy-modified electrically neutral ASO were located in
the inner aqueous space of neutral liposomes. This complex delivery
system with appropriate controls was used in the following
experiments to treat tumor in an experimental animal model of
ovarian carcinoma.
Histology of Tumor and Malignant Ascites
[0093] Solid primary tumors developed from cells obtained from
patients with malignant ascites demonstrated at least three
distinct histological types (FIG. 2, left panel). The predominant
type consisted of solid sheets and nests of markedly pleomorphic,
undifferentiated tumor cells with large, irregular nuclei and 1-2
prominent basophilic nucleoli. The cytoplasm was lightly
eosinophilic and cell margins were not identifiable. The tumor
cells displayed numerous mitotic figures, many of which were
atypical. Multinucleated tumor giant cells were common. The margins
of the tumor nests were surrounded by fine fibrovascular connective
tissue. The second distinct histological type, occupying
approximately 30% of the tumor, consisted of ribbons and nests of
well-differentiated tumor cells forming identifiable glands. The
epithelium of the glands was columnar, usually with a single row of
circular to elongated nuclei. The nuclei were moderately
pleomorphic and clear, with one or more nucleoli. The glandular
element showed a high level of mitotic activity. The third tumor
type consisted of aggregates of extremely large, pleomorphic cells
with multiple large basophilic nuclei. Many of these cells contain
abundant, brightly eosinophilic cytoplasm. The cytoplasm ranged in
consistency from dense to vacuolated. This phase of the tumor is
primarily localized to the margins of the tumor mass, adjacent to
the skeletal muscle and may represent a degenerative stage of the
cancer. There were suggestions of squamous differentiation,
including cytokeratin and keratin pearls. Infrequent psammoma
bodies were identified diffusely within the tumor mass. Psammoma
bodies are predominately seen in ovarian serous adenocarcinomas
(Pantanowitz et al. (2009) Acta Cytol., 53:263-267; Hiromura et al.
(2007) J. Comput. Assist. Tomogr., 31:490-492; Okada et al. (2005)
J. Nippon Med. Sch., 72:29-33).
[0094] Histologically untreated intraperitoneal metastases
consisted of numerous spherical tumor nodules adherent to white
adipose tissue (FIG. 2, middle panel). The nodules were comprised
primarily of poorly differentiated cancer cells, some displaying
several squamous characteristics. These cells were eosinophilic,
round to polygonal in shape, and demonstrated considerable nuclear
pleomorphism and a high mitotic rate. Many of the mitotic figures
were atypical, with tripolar or tetrapolar divisions easily
identified. In discrete regions, the cell membrane showed distinct
"prickle" attachments characteristic of squamous differentiation.
The tumor cell nuclei were large and showed variable amounts of
heterochromatin and 1-3 basophilic nucleoli. At the center of some
tumor nodules were cystic spaces containing a fibrovascular core.
The tumor nodules were enclosed by a layer of flattened to cuboidal
epithelial cells. The flattened cells showed clear squamous
differentiation, while the cuboidal cells were without discernable
lineage differentiation. The fat to which the nodules were attached
was focally infiltrated with polymorphonuclear neutrophilic
leukocytes and lymphocytes.
[0095] Histological analysis showed that mice with subcutaneous
tumors treated with combination of two DDS (LHRH-Lip-DOX-BCL2-MDR1
ASO and LHRH-Lip-CIS-BCL2-MDR1 ASO) underwent both apoptosis and
necrosis (FIG. 2, right panel). Many of the tumor nests displayed
central necrosis and glassy-eosinophilic cytoplasmic swelling.
Clusters of brightly eosinophilic tumor cells were noted; these
contained ample, swollen and degenerating cytoplasm with
well-developed zones of tumor necrosis. Treated tumor nodules
consisted of necrotic tumor tissues and showed a fibroblastic
reparative response. The majority of the nodules contained `ghosts`
of dead tumor cells, hyalinized connective tissue, and a mild mixed
inflammatory response. Only rare viable malignant cells were
identified within the nodules, and these were represented by single
or small clusters of cells, some of which are undergoing
degeneration. The degree of tumor killing was >99% of the cells.
Macrophages containing a granular, light gold material were present
within the dead tumor mass and abundantly within the lymph node. No
ascites development was registered in mice with primary tumor
treated with the aforementioned combination of DDS.
Expression of Targeted Genes and Proteins
[0096] Analysis of in vivo expression of targeted genes responsible
for cellular drug resistance showed that empty liposomes and LHRH
peptide alone (controls) did not change significantly the
expression of both MDR1 and BCL2 genes (FIGS. 3A and 3B, bars 1-3).
Treatment of mice bearing xenografts of human malignant ascites
with free DOX, CIS and their combination led to statistically
significant overexpression of both genes (FIGS. 3A and 3B, bars 4,
10, 16). Liposomal formulations of DOX and CIS induced
overexpression of BCL2 mRNA (FIG. 3B, bars 5, 11, 17). However, the
delivery of drugs as liposomal formulations led to the decrease in
the expression of the MDR1 mRNA when compared with free drugs (FIG.
3A, compare bars 4 and 5, 10 and 11, 16 and 17). Simultaneous
delivery of anticancer drugs with ASO targeted to MDR1 mRNA
decreased the expression of the MDR1 gene (FIG. 3A, bars 7-9,
13-15, 19-21). Similarly, simultaneous delivery of anticancer drugs
with ASO targeted to BCL2 mRNA decreased the expression of the BCL2
gene (FIG. 3B, bars 6, 8, 9, 12, 14, 15, 18, 20, 21). Direct
measurement of the expression of corresponding proteins (FIG. 4)
supports the results of gene expression analysis. In fact, the
expression of P-glycoprotein (encoded by the MDR1 gene) and BCL2
protein in xenografts of human ovarian cancers was increased after
the treatment of mice with free and liposomal forms of the drugs
(FIG. 4, panels a-b, #2-5), while incorporation of ASO into the
liposomal delivery system suppressed these proteins (FIG. 4, panels
a-b, #6-10).
[0097] It should be stressed that targeting of liposomal DDS to
tumor cells by LHRH peptide led to a more complete suppression of
the expression of both P-glycoprotein and BCL2 (FIG. 4, compare
#8-10 with #6-7 on panels a-b).
Apoptosis Induction
[0098] Apoptosis induction was studied in tumor, malignant ascites,
and healthy organs (liver, kidney, spleen, heart, lung and brain)
after the treatment of mice with different drug formulations by
measuring the expression of apoptosis executor--caspase 3 (FIG. 4,
panel c) and by immunochemical determination of histone-complexed
DNA fragments (mono- and oligonucleosomes) (FIG. 5). Empty
liposomes and LHRH did not induce detectable levels of apoptosis
either in tumor, ascites, or healthy organs (FIG. 5, bars 1, 2, 3).
Free DOX, CIS and their combination activated caspase 3 and induced
apoptosis in tumor and malignant ascites (FIG. 4, panel c, #2; FIG.
5, bars 1, 4, 10, 16). However, in addition to inducing apoptosis
in tumor and malignant ascites, treatment of mice with free drugs
led to the substantial apoptosis induction in the liver, kidney,
spleen, heart and lung. Incorporation of drugs into the liposomes
substantially enhanced their ability to induce apoptosis in solid
tumors and accompanying intraperitoneal metastases and limited
apoptosis induction in kidney, spleen, heart and lung (FIG. 5, bars
5, 11, 17). However, apoptosis in the liver remained augmented
after the treatment of mice with all non-targeted liposomal DDS
(FIG. 5, bars 5-8, 11-14, 17-20). Suppression of both types of
cellular resistance by ASO targeted to MDR1 and BCL2 mRNA
substantially increased the expression of caspase 3 (FIG. 4, panel
c, #6) and apoptosis induction (FIG. 5, bars 6-8, 12-14, 18-20).
Targeting of DDS containing anticancer drugs and suppressors of
pump and nonpump resistance specific to cancer cells by LHRH
peptide led to several positive consequences. First, it increased
the level of suppression of targeted proteins and therefore
augmented the activation of caspase 3 and apoptosis itself in
cancer cells (FIG. 4, panel c, #8-10; FIG. 5, bars 9, 15, 21).
Second, the delivery of drugs and other active components
specifically to cancer cells prevented the induction of apoptosis
in the liver as well as all other studied healthy organs (FIG. 5,
bars 9, 15, 21). Third, incorporation of a targeting moiety in DDS
led to the prevention of the development of intraperitoneal
metastases and malignant ascites (FIG. 5, bars 9, 15, 21 and FIG.
6, bars 9, 15, 21).
Antitumor Effect
[0099] The antitumor effect of all studied formulations was
estimated by the measurement of the volume of the primary tumor and
total mass of the intraperitoneal metastases. Treatment of mice
with saline, empty liposomes, and LHRH peptide (controls) did not
influence the growth of primary tumor or the development of
intraperitoneal metastases (FIG. 6, lines and bars 1-3). Free and
liposomal DOX slowed down the growth of the primary subcutaneous
tumor but was not effective in preventing the formation of
malignant ascites or intraperitoneal metastases and did not
statistically significantly decrease their mass (FIG. 6, lines and
bars 4, 5). Free and liposomal CIS was more effective in limiting
the growth of both primary tumor and malignant ascites when
compared to corresponding DOX formulations (compare lines and bars
10, 11 with 4, 5 in FIG. 6). The combination of two drugs (free and
liposomal forms) was more effective when compared with formulations
containing only one drug (compare lines and bars 16, 17 with 4, 5
and 10, 11 in FIG. 6). Inclusion of ASO targeted to MDR1 and/or
BCL2 mRNA into the liposomal drug formulations led to the more
significant suppression of the growth of the primary tumor,
carcinomatosis and ascites (FIG. 6, lines and bars 6-8, 12-14,
18-20). Targeting of DDS specifically to cancer cells by LHRH
peptide further increased antitumor activity of liposomal DDS and
prevented the development of intraperitoneal metastases and ascites
(FIG. 6, lines and bars 9, 15, 21).
[0100] Herein, the antitumor effect of a liposomal tumor-targeted
proapoptotic anticancer drug delivery system on the primary tumor
and intraperitoneal metastases was examined in a mouse model
developed after subcutaneous inoculation of cancer cells isolated
from malignant ascites of patients with ovarian carcinoma.
Inoculation of these cancer cells provoked, in untreated mice, the
development of two models of cancer. The first one represented a
subcutaneous model of aggressive primary tumor. The second model of
intraperitoneal metastases with ascites developed in approximately
80% of untreated mice. This mouse model of human intraperitoneal
carcinomatosis with ascites is different from traditionally used
orthotopic models when established cancer cells, primary tumor
isolates, or cells from human malignant ascites are
intraperitoneally injected into nude mice (De Cesare et al. (2010)
J. Immunother., 33:8-15; Hsieh et al. (2009) Cancer Sci.,
100:537-545; Pasquet et al. (2010) Int. J. Cancer 126:2090-2101;
Pourgholami et al. (2006) Clin. Cancer Res., 12:1928-1935; Song et
al. (2008) Cancer Biol. Ther., 7:76-80). This model more adequately
showed the natural progression of ovarian cancer with the
development of carcinomatosis and ascites. It is interesting that
this model can be effectively employed only when the cells that are
used for the initiation of primary subcutaneous tumor are very
invasive and multidrug resistant. It was found that intraperitoneal
metastases never developed when drug sensitive ovarian cancer cells
were injected subcutaneously. When established human multidrug
resistant ovarian cancer cell lines or primary tumor isolates were
used, malignant ascites accompanied only approximately 10% of
primary tumors. Consequently, one can conclude that cancer cells
isolated from human ovarian malignant ascites are substantially
more invasive when compared with established multidrug resistant
cancer cell lines. Such an assumption is also supported by the data
of Veatch et al. (Int. J. Cancer. (1994) 58:393-399) who found that
" . . . ascites cells were 4-fold more invasive than solid tumor
cells." In addition, the development of multidrug resistance
accompanied by overexpression of the MDR1 gene was observed in
tumors of patients treated with DOX (Abolhoda et al. (1999) Clin.
Cancer Res., 5:3352-3356). Taking into account that cells from
ovarian malignant ascites belong to a multidrug resistant
phenotype, effective therapy of intraperitoneal metastases and
ascites requires drugs with different mechanisms of action and the
simultaneous suppression of both cellular pump and nonpump
resistance. Ideally, any cytotoxic treatment should preferentially
target cancer cells sparing healthy tissues. This was successfully
accomplished by targeting the LHRH receptor (Chandna et al. (2007)
Mol. Pharm., 4:668-678; Dharap et al. (2005) Proc. Natl. Acad.
Sci., 102:12962-12967; Chandna et al. (2010) Pharm. Res.). The
experimental verification of this hypothesis showed several
advantages of the instant approach.
[0101] The first advantage is the simultaneous suppression of both
major types of cellular resistance. It was found that antisense
oligonucleotides targeted to MDR1 and BCL2 mRNA and delivered by
PEGylated liposomes effectively suppressed both the major drug
efflux pump (P-glycoprotein) and the antiapoptotic cellular defense
(BCL2 protein) leading to the inhibition of cellular pump and
nonpump resistance, respectively. Second, such suppression led to
the substantial enhancement of cell death and increased efficiency
of standard cytotoxic drugs to levels that could not be achieved
using conventional therapy by free or liposomal forms of the drugs.
Third, effective cell death induction in primary tumor and
intraperitoneal metastases led to the substantial regression of
tumor growth and decrease in total mass of intraperitoneal
metastases. Fourth, targeting of DDS specifically to ovarian cancer
cells significantly reduced the adverse side effects of the
treatment on healthy organs. This effect is explained by the
specific body distribution of the tumor targeted delivery system,
where the major part of intravenously injected tumor-targeted DDS
is accumulated in tumor cells, while only trace amounts of DDS can
be found in healthy organs (Chandna et al. (2007) Mol. Pharm.,
4:668-678; Dharap et al. (2005) Proc. Natl. Acad. Sci.,
102:12962-12967). The fifth advantage of the instant approach
includes the prevention of the development of detectable
intraperitoneal metastases and ascites after the treatment of an
aggressive primary ovarian tumor with the targeted DDS. Moreover,
it was found that each targeted DDS containing either one drug
alone or a drug combination prevented the formation of
carcinomatosis and ascites. In contrast, similar non-targeted
systems containing the same components (drug, ASO targeted to MDR1
and ASO targeted to BCL2 mRNA) did not produce such an effect.
These results reinforce the importance of targeting anticancer
therapy specifically to cancer cells (by a ligand to extracellular
receptors overexpressed in cancer cells) for the prevention of the
development of intraperitoneal metastases and ascites. The
aforementioned advantages of the proposed targeted combinatorial
treatment of primary aggressive ovarian tumor and prevention of the
development of intraperitoneal metastases make the instant approach
of utilizing tumor-targeted delivery systems ideal for clinical
applications.
Example 2
[0102] Novel therapeutic approaches based on RNA interference
(RNAi), a post-transcriptional gene silencing mechanism, mediated
by small duplex RNA, attract substantial attention (Gary et al.
(2007) J. Control Rel., 121:64-73; Patil et al. (2008) Bioconjug.
Chem., 19:1396-1403; Patil et al. (2009) Biomacromol., 10:258-266;
Saad et al. (2008) Nanomed., 3:761-776; Taratula et al. (2009) J.
Control Rel., 140:284-293; Zhou et al. (2006) Chem. Commun. (Carob)
22:2362-2364). However, similar to other gene therapy strategies,
low stability in the bloodstream and poor cell penetration ability
of naked small interfering RNA (siRNA) represent main obstacles for
the practical use of these methodologies (Gary et al. (2007) J.
Control Rel., 121:64-73; Kang et al. (2005) Pharm. Res.,
22:2099-2106; Patil et al. (2008) Bioconjug. Chem., 19:1396-1403;
Patil et al. (2009) Biomacromol., 10:258-266; Taratula et al.
(2009) J. Control Rel., 140:284-293; Zhou et al. (2006) Chem.
Commun. (Carob) 22:2362-2364). Viruses developed the ability to
deliver double and single stranded DNA and RNA across the cell
membrane; however, the immune response elicited by viral proteins
limited their use as delivery agents (Bessis et al. (2004) Gene
Ther., 11:S10-17). Therefore, the development of non-viral systems,
which are able to protect siRNA during its journey in the
circulation to the site of action and effectively deliver it across
the cell membrane to the cytoplasm to guide the sequence-specific
mRNA degradation, is very important in order to exploit their
therapeutics potential.
[0103] Studies of non-viral gene therapy based on plasmid DNA
(pDNA) have being carried out for years to improve systemic
delivery and transfection efficiencies of pDNA to the levels
required for in vivo clinical trials. It has been recognized that a
prerequisite for the facile transport of pDNA through the cell
membrane is the condensation (packaging) of the nucleic acids into
nanoparticles, which can protect pDNA by sterically blocking its
degradation by nucleolytic enzymes (Vijayanathan et al. (2002)
Biochem., 41:14085-14094). pDNA and siRNA are both double-stranded
nucleic acids, they have anionic phosphodiester backbones with the
same negative charge to nucleotide ratio, and can interact
electrostatically with cationic agents (Gary et al. (2007) J.
Control Rel., 121:64-73). Therefore, one can use the knowledge
gained from the longer-studied pDNA to develop systems for an
effective delivery of siRNA. However, despite the similarities,
pDNA and siRNA are very different from each other in molecular
weight and molecular topography with potentially important
consequences. The pDNA used in gene therapy is often several kilo
base pairs long and possesses a molecular topography, which allows
the condensation into small, nanometre size particles when
complexed with a cationic agent (Gary et al. (2007) J. Control
Rel., 121:64-73; Spagnou et al. (2004) Biochem., 43:13348-13356).
Unlike pDNA, the persistence length (the length scale over which
the chains behave as rigid rods) of double-stranded RNA is
.about.70 nm (Kebbekus et al. (1995) Biochem., 34:4354-4357) which
is about 260 base pairs (bp) based on the value of 2.7 .ANG./bp
(Shah et al. (1999) J. Mol. Biol., 285:1577-1588). Therefore, siRNA
with 21 bp essentially should behave as a rigid rod and is
difficult to bend. Consequently, the different interactions with
cationic agents may result in undesirably large complexes or
incomplete encapsulation of siRNA molecules, which thereby may
expose siRNA to potential enzymatic or physical degradation in the
bloodstream prior to the delivery to the targeted cells (Spagnou et
al. (2004) Biochem., 43:13348-13356).
[0104] Over the years, cationic liposomes and polycations have been
explored as nonviral vectors for the delivery of siRNA (Garbuzenko
et al. (2009) Pharm. Res., 26:382-394; Saad et al. (2008) Nanomed,
3:761-776). An advantage of using polycationic polymers, such as
Poly(Ethyleneimine) (PEI), is that they allow for an efficient gene
transfer without the need for agents facilitating endosomal escape
of the payload. However, such polymers have a wide range of
molecular weight distribution and their transfection efficiency and
cytotoxicity are dependent on the molecular weight and
polydispersity (Gary et al. (2007) J. Control Rel., 121:64-73; Pack
et al. (2005) Nat. Rev. Drug Discov., 4:581-593; Spagnou et al.
(2004) Biochem., 43:13348-13356; Vijayanathan et al. (2002)
Biochem., 41:14085-14094). In contrast, the novel highly branched
three-dimensional molecules, called dendrimers, have defined
molecular weight and a large number of controllable surface charges
and surface functionalities (Dufes et al. (2005) Adv. Drug Deliv.
Rev., 57:2177-2202). These properties of dendrimers provide a
platform for an effective siRNA intracellular delivery, with
potentially less complications from heterogeneity and variable
chemistry, commonly seen in other nonviral vectors, such as
cationic lipids and PEI (Chen et al. (2006) Nanotechnol.,
17:5449-5460; Santhakumaran et al. (2004) Nucl. Acids Res.,
32:2102-2112; Vijayanathan et al. (2002) Biochem., 41:14085-14094).
Poly(propyleneimine) (PPI) dendrimers are members of a class of
amine-terminated polymers that have been used as efficient gene
delivery vectors with low cytotoxicity in a wide range of mammalian
cell lines (Chen et al. (2006) Nanotechnol., 17:5449-5460;
Santhakumaran et al. (2004) Nucl. Acids Res., 32:2102-2112;
Taratula et al. (2009) J. Control Rel., 140:284-293). The multiple
functional groups on the surface of these dendrimers also allow the
design of multifunctional delivery systems containing other active
components (e.g. targeting moieties, etc.) in addition to siRNA
(Patil et al. (2008) Bioconjug. Chem., 19:1396-1403; Taratula et
al. (2009) J. Control Rel., 140:284-293). However, most of the
synthetic vectors, including dendrimers, usually possess lower
efficiency in intracellular nucleic acid delivery (Pack et al.
(2005) Nat. Rev. Drug Discov., 4:581-593). Therefore, fundamental
understanding of the molecular structure of nonviral vectors and
their structure-function relationship is essential for rational
design of nonviral delivery vehicles with the therapeutic
effectiveness that can match or be better than the viral
counterpart. Consequently, by linking the chemical structures of
cationic vehicles to the morphology and physicochemistry of the
respective nucleic acid complexes and their biological properties
on cellular and systemic levels is essential for the development of
nonviral delivery vehicles with efficiency that can match or be
better than their viral counterpart. The present work is aimed at
studying the properties of nanoparticles formed by the complexation
of model siRNA with PPI dendrimers of different generations, the
morphology and cellular toxicity of such complexes and their
efficiency to deliver the payload into the cytoplasm and silence
the targeted gene.
Material and Methods
Materials
[0105] Dendrimers, poly(propyleneimine) octaamine (DAB Am-8,
generation-2, PPI G2), poly(propyleneimine) hexadecaamine (DAB
Am-16, generation-3, PPI G3), poly(propyleneimine) dotriacontaamine
(DAB Am-32, generation-4, PPI G4), and poly(propyleneimine)
tetrahexacontaamine, DAB-Am-64, generation-5, PPI G5) were
purchased from Aldrich (Milwaukee, Wis.), and used without further
purification. Ethidium Bromide (EtBr) solution was purchased from
Promega (Madison, Wis.). The sequence of siRNA targeted to BCL2
mRNA (custom synthesized by Ambion, Austin, Tex.), was
5'-GUGAAGUCAACAUGCCUGC-dTdT-3' (sense strand; SEQ ID NO: 4) and
5'-GCAGGCAUGUUGACUUCAC-dTdT-3' (antisense strand; SEQ ID NO: 5).
6-FAM siRNA (SiGLO.RTM. Green) was obtained from Applied Biosystems
(Ambion, Inc., Foster City, Calif.). All other chemicals were
purchased from Fisher Scientific (Fairlawn, N.J.).
Cell Line
[0106] Human A549 lung carcinoma cells were obtained from the ATTC
(Manassas, Va., USA). Cells were cultured in RPMI 1640 medium
(Sigma, St. Louis, Mo.) supplemented with 10% fetal bovine serum
(Fisher Scientific, Fairlawn, N.J.). Cells were grown at 37.degree.
C. in a humidified atmosphere of 5% CO.sub.2 (v/v) in air. All
experiments were performed on cells in the exponential growth
phase.
Ethidium Bromide (EtBr) Dye Displacement Assay
[0107] Fluorescence titration of siRNA-ethidium bromide complexes
with different generations of PPI dendrimers were performed as
follows. The complexes prepared from siRNA with EtBr intercalated
at 4:1 ratio in water and 1 .mu.L aliquots of the dendrimer
solutions were sequentially added to 2 .mu.M solution of siRNA in
180 .mu.L of water containing EtBr. After each addition, the
mixture was stirred and the fluorescence of the solution was
measured (490 nm excitation; 590 nm emission). The total dendrimer
amount added to the siRNA solution exceed 5% of the total volume of
the mixture, hence sample dilution factors on the measured
fluorescence emission intensity was corrected. All fluorescence
measurements were performed using a Cary-Eclipse fluorescence
spectrophotometer (Varian, Inc, Palo Alto, Calif.). The relative
fluorescence was based on three independent experiments and
calculated using the following equation: % Relative
Fl=[(Fl(obs)-Fl(EtBr))/(Fl(siRNA+EtBr)-Fl(EtBr))].times.100, where
Fl (obs)-fluorescence of siRNA+ethidium bromide+complexation agent;
Fl (EtBr)-fluorescence of ethidium bromide alone;
Fl(siRNA+EtBr)-fluorescence of siRNA+ethidium bromide.
Formulation of siRNA-Dendrimer Complexes
[0108] The complexes of siRNA with each generation of PPI
dendrimers (G2, G3, G4, and G5) were designed at constant
amine/phosphate ratios (N/P ratio) such as 2.4. Briefly, 100 .mu.M
siRNA solutions were mixed with deionized water and an appropriate
amount of the dendrimers was added. The final concentration of
siRNA in the solution was 4.0 .mu.M, while the concentrations of
PPI G2, G3, G4, and G5 dendrimers were 50.4 .mu.M, 25.2 .mu.M, 12.6
.mu.M, and 6.3 .mu.M, respectively. The complexes were stirred and
equilibrated for 30 minutes prior to analysis.
Atomic Force Microscopy (AFM)
[0109] In order to obtain AFM images of formulated complexes, 5
.mu.L aliquots of siRNA-dendrimer solutions were deposited on a
freshly cleaved mica surface. After 5 minutes of incubation, the
surface was rinsed with several drops of nanopure water (distilled
water filtered through an ion filter with organic compounds also
has been removed using a carbon filter resulting in analytical
grade water), and dried under a flow of dry nitrogen. AFM images
were obtained using Nanoscope IIIA AFM (Digital Instruments, Santa
Barbara, Calif.) in tapping mode, operating in an ambient
atmosphere.
Agarose Gel Retardation Assay
[0110] The complexes of siRNA with different generations of PPI
dendrimers were prepared in water as already described. Free siRNA
was used as the control. Double-stranded RNA ladder (New England
Biolabs) with the smallest base pairs at 21 was used as a size
reference. The samples were further diluted with water and
electrophoresed in 4% agarose gel at 100 mV for 60 minutes in TBE
buffer and stained with EtBr. The gels were digitally photographed,
and scanned using Gel Documentation System 920 (NucleoTech, San
Mateo, Calif.).
Dynamic Light Scattering (DLS)
[0111] The DLS studies were performed using the Dawn EOS
multi-angle light scattering spectrometer modified with a QELS
attachment (Wyatt Technology Corp., Santa Barbara, Calif.). Data
were collected at an angle of 108.degree. using an avalanche
photodiode and an optical fiber and processed with the Wyatt QELS
software (regularization analysis). Each light scattering
experiment consisted of 5 or more 60 second independent
readings.
In Vitro Cytotoxicity
[0112] A modified MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay was used to assess the cytotoxicity of different generations
of PPI dendrimer as previously described (Jayant et al. (2007)
Pharm. Res., 24:2120-2130). To measure cytotoxicity, human A549
lung carcinoma cells were separately incubated in a microtiter
plate with different concentrations of each PPI G2, G3, G4, and G5
dendrimers. Control cells received an equivalent volume of fresh
medium. The duration of incubation was 24 hours. On the basis of
these measurements, cellular viability was calculated for each
dendrimer concentration. A decrease in the cellular viability
indicated an increase in dendrimer toxicity.
Cellular Internalization
[0113] To analyze cellular internalization and intracellular
localization of the condensed complexes, 6-FAM labelled siRNA was
used as previously described (Saad et al. (2008) J. Control Rel.,
130:107-114; Taratula et al. (2009) J. Control Rel., 140:284-293).
Prior to the visualization, A549 cells were plated (20,000 cells
per well) in a 6-well tissue culture plate. The cells were treated
within 24 hours with the siRNA complex prepared with different
generations of PPI dendrimer. The treatment of the A549 cells was
performed in such a manner that the final concentration of siRNA
was 0.25 .mu.M and the concentrations of PPI G2, G3, G4, and G5
dendrimers were 3.15 .mu.M, 1.58 .mu.M, 0.79 .mu.M, and 0.39 .mu.M,
respectively. After 24 hours of treatment, the cells were washed
three times with Phosphate Buffered Saline (PBS) and 1 mL of media
was added to each well. Cellular internalization of siRNA-dendrimer
complexes were analyzed by fluorescent (Olympus America Inc.,
Melville, N.Y.) and confocal (Leica Microsystems Inc., Bannockburn,
Ill.) microscopes. To obtain intracellular distribution of siRNA,
ten optical sections, known as a z-series, were photographed
sequentially, by confocal microscope, along the vertical (z) axis
from the top to the bottom of the cell. The obtained fluorescent
images were digitally scanned and fluorescence inside cells
(reflecting intracellular accumulation of labelled siRNA) was
expressed in arbitrary units.
Gene Expression
[0114] Quantitative reverse transcription-polymerase chain reaction
(RT-PCR) was used for the analysis of expression of genes encoding
BCL2 protein and .beta..sub.2-microglobulin (internal standard) as
previously described (Pakunlu et al. (2003) Pharm. Res.,
20:351-359). RNA was isolated after 24 hours incubation of cancer
cells with siRNA-dendrimer complexes, using an RNeasy kit (Qiagen,
Valencia, Calif.). First strand complementary DNA (cDNA) was
synthesized by Ready-To-Go You-Prime First-Strand Beads (Amersham
Biosciences, Piscataway, N.J.) with 4 mg of total cellular RNA and
100 ng of random hexadeoxynucleotide primer (Amersham Bioscience,
Piscataway, N.J.). After synthesis, the reaction mixture was
immediately subjected to PCR, which was carried out using
GenAmp.RTM. PCR System 2400 (Perkin-Elmer, Shelton, Conn.). The
following pairs of primers were used: BCL2--GGA TTG TGG CCT TCT TTG
AG (sense; SEQ ID NO: 6), CCA AAC TGA GCA GAG TCT TC (antisense;
SEQ ID NO: 7); .beta..sub.2-microglobulin (.beta..sub.2-m)--ACC CCC
ACT GAA AAA GAT GA (sense; SEQ ID NO: 8), ATC TTC AAA CCT CCA TGA
TG (antisense; SEQ ID NO: 9). PCR products were separated in 4%
NuSieve.RTM. 3:1 Reliant agarose gels in 1*TBE buffer (0.089 M
Tris/Borate, 0.002 M EDTA, pH 8.3; Research Organic Inc., Cleveland
Ohio) by submarine gel electrophoresis. The gels were stained with
ethidium bromide, digitally photographed and scanned using Gel
Documentation System 920 (NucleoTech, San Mateo, Calif.). Gene
expression was calculated as the ratio of mean band intensity of
analyzed RT-PCR product (BCL2) to that of the internal standard
(.beta..sub.2-m). The value of BCL2 gene expression for the cells
incubated with fresh medium (control) was set as 100%.
Statistical Analysis
[0115] Data were analyzed as described hereinabove in Example I for
four to eight independent measurements.
Results
[0116] Evaluation of Interaction Between siRNA and PPI Dendrimers
of Different Generation by EtBr Displacement Assay
[0117] Similarly to pDNA, the complex formation between negatively
charged siRNAs and positively charged polycations is primarily
driven due to electrostatic interaction. The amount of polycations
needed for the complex formation could be estimated by N/P
(nitrogen/phosphate) ratios, which refer to the ratio of the
positively charged primary amine groups of polycations to
negatively charged phosphate groups of siRNA (Gary et al. (2007) J.
Control Rel., 121:64-73). EtBr intercalates between the base pairs
of the DNA double helix, yielding a highly fluorescent DNA-EtBr
complex (Izumrudov et al. (1999) Biopolymers, 52:94-108). Upon
polycations binding, the DNA double helix structure is distorted
and EtBr is expelled from the DNA-EtBr complex, resulting in a
decrease of fluorescence (Izumrudov et al. (1999) Biopolymers,
52:94-108). The degree of EtBr displacement thus provides for a
measure of the binding affinity, indicating the relative strength
of the interaction between DNA and polycations. A similar process
was found for siRNA in the present study, as shown in FIG. 7A.
Although the exact mechanism of EtBr release from siRNA requires an
additional detailed study, the differences in the ability of
dendrimers to displace EtBr from siRNA provide a comparison of the
binding affinities between siRNA and the PPI dendrimers of
different generations (Thomas et al. (2005) Proc. Natl. Acad. Sci.,
102:5679-5684). FIG. 7B shows that fluorescence intensities
decreased progressively with increasing N/P ratios up to 0.36,
0.55, 0.74, and 0.92 for PPI G4, G5, G3 and G2, respectively. These
ratios, which represent the apparent end point of complexation for
each PPI dendrimer generation, respectively, indicated that the
interaction strength between PPI dendrimers and siRNA decreases as
follows: G4>G5>G3>G2.
Size and Morphology of Formulated siRNA-Dendrimer Complexes
[0118] Tapping mode AFM was used to study the size and morphology
of siRNA complexes prepared in the presence of different
generations of PPI dendrimers. To ensure a complete siRNA
complexation, all the siRNA complexes with different generation
dendrimers were formulated at a ratio of 2.4, which is several
times higher than the apparent end points of condensation observed
by EtBr dye displacement assay. Agarose gel retardation assay was
applied to further ensure that this N/P ratio was sufficient for
siRNA complex formation (FIG. 8A). As shown in FIG. 9, fiber- and
sphere-shaped nanostructures were formed in the presence of PPI G2
to G5 dendrimers after 30 minutes of condensation. The lower
generations of dendrimers are capable to complex siRNA resulting in
the formation of randomly aggregated nanofibers (G2) (FIG. 9A), or
a mixture of nanofibers and toroids (doughnut-shaped objects) (G3),
respectively (FIG. 9B). The height of the fiber-like structures was
around 1.7 nm with the length varied from 100 nm to several .mu.m,
whereas the average height and diameter of toroids were 2.9.+-.0.2
nm and 176.6.+-.12.3 nm, respectively. The relatively weak
electrostatic cooperative interaction between siRNA and PPI G2 or
PPI G3 may be the reason to form this fiber shaped structures. On
the other hand, the products of siRNA condensation with higher
generations of PPI dendrimers appear to be relatively uniform
nanoparticles with average diameters 150.3.+-.24.7 nm and
150.1.+-.22.2 nm for PPI G4 and PPI G5, respectively (FIG. 9C-D).
Whereas the average heights of the siRNA complexes formed in the
presence of PPI G4 and PPI G5 were 6.2.+-.0.9 nm and 5.4.+-.0.9 nm,
respectively. It is worthy to mention that the height values of
spherical gene delivery complexes measured by AFM are almost always
lower than their diameters. The major factors contributing to the
lower height values are elastic deformation induced by AFM
tip-sample interaction and the compression caused by the attractive
forces between complexes and the substrate (Oh et al. (2006) J. Am.
Chem. Soc., 128:5578-5584). DLS technique was also used to
determine the hydrodynamic size of the siRNA complexes without
perturbation by the surface immobilization process and the AFM tip.
Similar trend was found with increasing generations of the PPI
dendrimer, even though the absolute size of the complexes measured
by DLS was relatively small. The hydrodynamic diameter for siRNA
complexes formed in the presence of higher generations PPI
dendrimers were 96.8.+-.11.3 nm and 101.2.+-.23.8 nm for PPI G4 and
PPI G5, respectively. Additionally, the solutions of siRNA-PPI G2
and siRNA-PPI G3 had hydrodynamic diameters of 224.0.+-.58.5 nm and
129.8.+-.26.4 nm, which was attributed to the fiber and torus-like
constructions (FIG. 10A).
Cellular Internalization and Intracellular Localization of
siRNA-Dendrimer Complexes
[0119] To achieve an enhanced gene silencing therapeutic effect,
sequence-specific siRNA molecules have to be delivered efficiently
into the cytoplasm of cells, where the RNAi machinery is located.
To determine whether the studied PPI dendrimers were able to
efficiently deliver siRNA into cancer cells, the cellular
internalization of the FAM-labelled siRNA (free or complexed) was
investigated by fluorescence microscopy after their incubation with
A549 human lung cancer cells. The quantitative evaluation of siRNA
internalization efficiency based on the emission intensity showed
that the intracellular uptake of the complexed and free siRNA was
dramatically different and strongly dependent on dendrimer
generation (FIG. 10B). Whereas free siRNA as well as siRNA-PPI G2
complexes were not internalized by the cells, siRNA complexes
formulated in the presence of G3, G4 and G5 dendrimers were capable
of facilitating siRNA internalization into the cytoplasm. Overall,
the efficiency of the studied complexation agents to provoke siRNA
intracellular uptake declines in the following order: PPI G4>PPI
G5>PPI G3>PPI G2 (FIG. 10B). The result is consistent with
the ability of the dendrimers in provoking siRNA condensation into
discrete nanoparticles. In the present study, the intracellular
distribution of the siRNA-PPI complexes in A549 human lung cancer
cells was investigated by confocal microscopy (FIG. 11). Analysis
of the obtained images demonstrated the fluorescence from 6-FAM
labelled siRNA delivered by PPI G3, G4 and G5 dendrimers was
localized primarily in the cytoplasm of the cells and was not
registered in the nuclei. The siRNA complexes formed with PPI G3-G5
dendrimers were uniformly distributed in the cytoplasm from the top
to the bottom of the cells (FIG. 11C-E).
Gene Silencing Efficiency of siRNA-Dendrimer Complexes In Vitro
[0120] In order to achieve therapeutic effects by the internalized
siRNA, the complex which protects siRNA in an extracellular
environment has to be able to escape from the endosome and release
siRNA into the cytoplasm. To evaluate the silencing efficacy of the
siRNA delivered by PPI dendrimers, the siRNA complexes formulated
in the presence of four different generations of PPI dendrimers
were incubated with A549 lung cancer cells and the expression of
targeted BCL2 mRNA was measured by RT-PCR. FIG. 12 shows that siRNA
delivered by PPI G5 and G4 dendrimers sufficiently induced
degradation of the targeted BCL2 mRNA. siRNA-PPI G5 complexes
resulted in 75% gene knockdown, while siRNA-PPI G4 exhibited almost
complete suppression of the targeted BCL2 mRNA in A549 lung cancer
cells. On the other hand, siRNA complexed with PPI G3 dendrimer
resulted in only a 40% decrease in gene knockdown and the condensed
siRNA with PPI G2 dendrimer showed no statistically significant
silencing effect. Gene silencing was not observed with naked siRNA
and all generations of PPI dendrimer without siRNA.
In Vitro Cytotoxicity of PPI Dendrimers
[0121] Cytotoxicities of gene transfection vectors including viral
vectors, inorganic nanoparticles, cationic liposomes and polymers
are considered as a substantial concern for their clinical
applications. In order to examine whether different generations of
PPI dendrimers influenced cell viability of A549 human lung cancer
cells, the MTT assay was employed. FIG. 8B shows the average data
obtained in three independent experiments with increasing
concentration of PPI dendrimers of different generations. The
cytotoxicity of the dendrimers increased with the generation,
following the order of the least to most cytotoxic:
G2<G3<G4<G5. The data are correlated with membrane damage
effects, implying that the increase in positive charge causes more
efficient binding to the negatively charged cell membrane and
destabilizing them (Kunath et al. (2003) J. Control Rel.,
88:159-172). In the in vitro experiments described here, the
concentrations of PPI G2, G3, G4 and G5 dendrimers was 3.15 .mu.M,
1.58 .mu.M, 0.79 .mu.M and 0.39 .mu.M, respectively, which are
substantially lower than the threshold of cellular toxicity of the
carriers.
Antitumor Activity of PPI Dendrimer
[0122] The silencing of BCL2 and antitumor activity of
LHRH-Targeted and Non-Targeted PPI Dendrimer formulations
containing cisplatin and siRNA were determined. Briefly, mice (see
mouse xenograft model above) were treated three timed within 14
days with the following formulations: (1) Control (saline); (2) PPI
dendrimer; (3) LHRH; (4) Naked siRNA targeted to BCL2 mRNA; (5)
BCL2 siRNA-PPI-DTBP-PEG; (6) LHRH-BCL2 siRNA-PPI-DTBP-PEG; (7) Free
CIS; (8) BCL2 siRNA-PPI-DTBP-PEG+CIS; (9) LHRH-BCL2
siRNA-PPI-DTBP-PEG+CIS. The results are presented in FIG. 13.
Notably, the administration of LHRH targeted dendrimers with BCL2
siRNA and cisplatin lead to the greatest reduction of tumor
volume.
[0123] Here, PPI dendrimers of different generations (G2 to G5)
were evaluated in terms of their ability for siRNA condensation
ability and intracellular delivery of siRNA. It was found that the
structure of dendrimers substantially influences their abilities to
form stable complexes with siRNA and to deliver the payload to the
cytoplasm. By evaluating the results from all experiments, PPI G4
dendrimer is the most suitable for this purpose.
[0124] In general, the binding affinity of polycations to nucleic
acids is determined by the number of charges and their density per
molecule available for the interaction. With each increasing
generation of the PPI dendrimer, the number of surface amine
groups, which are most likely to bind siRNA, doubles. Therefore, it
can be assumed that the strength of the bond increases with
dendrimer generations, which is largely due to the increase in the
number of primary amino groups in higher generation dendrimers.
Additionally, it was reported that DNA binding affinity with
dendrimers was largely affected by the size of dendrimers, which
increases with each generation (Dufes et al. (2005) Adv. Drug
Deliv. Rev., 57:2177-2202). Molecular modelling studies of PPI
dendrimers of all 5 generations (Zinselmeyer et al. (2002) Pharm.
Res., 19:960-967) showed that the PPI G1 appears to bind across the
major groove of DNA, PPI G3 is sufficiently large to bind across an
entire helical turn, spanning major as well as minor grooves. For
generation 4 and 5, a significant proportion of the PPI dendrimer
molecules were able to interact directly with multiple DNA
strands.
[0125] Here, it was found that PPI G4 was more efficient in terms
of complex formation with siRNA, despite the PPI G5 dendrimer
containing more primary amino groups (64 vs. 32) and have a larger
size (the hydrodynamic diameters of G4 and G5 dendrimers in water
solutions are about 3.12 and 3.96 nm, respectively (Rietveld et al.
(1999) Macromolecules, 32:4608-4614)). These results are in a good
agreement with other studies (Santhakumaran et al. (2004) Nucleic
Acids Res., 32:2102-2112), which demonstrated that PPI G4 dendrimer
was the most efficient triplex-forming oligodeoxynucleotide
delivery agent for five cancer cell lines. It is also consistent
with the previous study on the compaction and delivery of antisense
oligonucleotides into breast cancer cells (Chen et al. (2006)
Nanotechnol., 17:5449-5460).
[0126] The better efficiency of PPI G4 dendrimer compared to PPI G5
dendrimer in provoking siRNA complexation could be explained by the
difference in the flexibility of the dendrimers, which decreases
with an increase in the number of dendrimer generation
(Guillot-Nieckowski, et al. (2007) New J. Chem., 31:1111-1127). It
was reported that partially degraded PAMAM dendrimers were more
efficient than the intact dendrimers in condensation of DNA and
antisense oligonucleotides, because they are more easily collapsed
after the neutralization of the charge with nucleic acid due to the
enhanced flexible structures (Dennig et al. (2002) J. Biotechnol.,
90:339-347.).
[0127] It was found that G4 dendrimer has the highest cooperative
electrostatic interaction with the siRNA despite G5 dendrimer has
the highest charge density, possibly due to the decreased
flexibility or steric hindrances of G5 that are resulted in a
relatively lower cooperative interaction with the siRNA. Since the
flexibility of a transfection vector as well as the size and the
amount of protonated primary amines on the outer surface drives its
ability to compact nucleic acids tightly, the optimal combination
in PPI G4 structures makes it the most efficient complexation agent
among all other generations.
[0128] The size and morphology of siRNA complexes are the important
factors in siRNA internalization and can dramatically influence
their transfection efficiency (Chen et al. (2006) Nanotechnol.,
17:5449-5460; Patil et al. (2008) Bioconjug. Chem., 19:1396-1403;
Patil et al. (2009) Biomacromolecules, 10:258-266.; Taratula et al.
(2009) J. Control Release, 140:284-293). It was found that lower
generations of dendrimers (G2 and G3) formed with siRNA randomly
aggregated nanofibers (G2) or a mixture of nanofibers and toroids
(G3). In contrast, higher generations of PPI dendrimers formed
well-condensed relatively uniform spherical nanoparticles. At the
same time, it was revealed that complexes of G2 and G3 dendrimers
with siRNA were unable to penetrate cellular cytoplasm in vitro.
Relatively low cellular internalization of siRNA delivered by PPI
G2 and PPI G3 dendrimers may be at least partially related to the
presence of large aggregated structures (fibers and thin toroids)
in the complexes. Furthermore, it was reported that electrostatic
interactions between the negatively charged cell membranes and
positively charged particles can enhance their cellular uptake
(Chen et al. (2006) Nanotechnol., 17:5449-5460). It was reported
that complexation of DNA with PPI dendrimers G1 and G2 led to the
formation of electroneutral complexes even at dendrimer:DNA charge
ratios >1 (Kabanov et-al. (2000) Macromolecules, 33:9587-9593).
The higher generations of dendrimers were able to produce charged
soluble complexes because of the ability to form overstoichiometric
complexes with a net positive charge. Recently, it was also found
that all five generations of PPI dendrimers could provoke
nanoparticles formation with Antisense Oligonucleotides (ASO)
targeted to the c-myc oncogene (Chen et al. (2006) Nanotechnol.,
17:5449-5460). However, only generation 4 and 5 dendrimers could
deliver ASO to cell nuclei as determined from a confocal
microscopic study. Zeta potential measurement of the ASO-PPI
complexes formed with dendrimers of different generations shows
that the complexes formed from higher generation PPI dendrimers had
much higher positive zeta potentials than the lower generation
dendrimers. Even though agarose gel retardation assay demonstrated
that the siRNA-PPI dendrimer complexes formed from all five
generations were retarded, the complexes with lower generation
dendrimers may also have lower positive charges, similar to the
situation with ASO-dendrimer complexes. Additionally, it was found
(Liu et al. (2001) J. Biol. Chem., 276:34379-34387) that large DNA
aggregates formed under non-cooperative conditions could not be
internalized into cells, and only the fully compacted DNA
nanoparticles formed from cooperative binding were able to
penetrate through the cellular plasma membrane. Since the PPI G5
dendrimer has less cooperative electrostatic interaction with siRNA
when compared with G4, it was hypothesized that the siRNA
nanoparticles formed from these two generations may have different
physical chemical properties, which may influence the cell uptake.
It is also possible that the amount of siRNA nanoparticles formed
with PPI G5 dendrimers via cooperative electrostatic binding was
less than that in G4 dendrimers. In addition, it has recently been
demonstrated (Taratula et al. (2009) J. Control Release,
140:284-293) that certain siRNA-PPI G5 nanoparticles showed
aggregation in cell medium, which could prevent some portion of
siRNA from the cellular internalization. Although further, more
detailed studies are required for the investigation of detailed
mechanisms of the phenomenon, one can conclude that generation 4
and 5 dendrimers, particularly G4, provide the most efficient
intracellular delivery of complexated siRNA.
[0129] Herein, it was found that siRNA delivered by dendrimers
located mainly in the cellular cytoplasm but not in nuclei. Since
siRNA functions by binding to RNA-induced silencing complex in the
cytoplasm, the delivery of siRNA by PPI dendrimers to the cytoplasm
but not to nucleus may have the advantage of increasing siRNA gene
silencing activity and avoiding toxicity to the nucleus. It has
been previously reported that efficiency of RNAi activity was
dependent on the siRNA localization in different intracellular
compartments (Chiu et al. (2004) Chem. Biol., 11:1165-1175). Most
of the reports show that siRNA delivered by both liposome and
cationic polymers is localized in cytoplasm and not in the nucleus
even after extended periods of time (Garbuzenko et al. (2009)
Pharm. Res., 26:382-394.; Keller, M. (2005) J. Control Release,
103:537-540; Saad et al. (2008) Nanomed., 3:761-776; Spagnou et al.
(2004) Biochem., 43:13348-13356). However, it was reported that
PAMAM dendrimers have a tendency to alter siRNA subcellular
localization pattern, which is concentration dependent (Chiu et al.
(2004) Chem. Biol., 11:1165-1175). Using higher concentration of
PAMAM, they observed that the internalized siRNA was localized in
both nucleus and cytoplasm. The authors believe that such
distribution correlates with the observed lower RNAi activity (Chiu
et al. (2004) Chem. Biol., 11:1165-1175). Overall, the present
study showed that the targeted gene silencing ability of the
siRNA-dendrimer complex depends on the dendrimer generation,
following the same trend as the ability of PPI dendrimer in
provoking siRNA complexation and facilitating cellular
internalization.
[0130] It was shown that PPI dendrimers can be used for the
delivery of a plasmid DNA (Zinselmeyer et al. (2002) Pharm. Res.,
19:960-967). The greater conformational mobility of the long DNA
molecules may facilitate its interaction with the extended
structure of lower generation dendrimers more efficiently than the
interaction of these dendrimers with the 21 bp siRNA in this study.
They also found that the number of binding sites between DNA and
the dendrimer increased with molecular weight. It is possible that
for a long DNA molecule, sufficient binding sites may be achieved
with lower generation dendrimers compared with small siRNA. This is
consistent with the reports showing that short DNA molecules were
more difficult to condense into well-defined nanoparticles
(Bloomfield, 1996; Zinselmeyer, et al., 2002). Due to
physicochemical and structural differences, conditions for
transfection of plasmid DNA could be different from conditions for
transfection of siRNA. The conditions may be more comparable to the
short antisense and triplexing forming oligonucleotides (Chen et
al. (2006) Nanotechnol., 17:5449-5460; Santhakumaran et al. (2004)
Nucleic Acids Res., 32:2102-2112).
[0131] Among all dendritic vectors, PAMAM are the most extensively
used carriers for plasmid DNA and antisense oligonucleotides (Kang
et al. (2005) Pharm. Res., 22:2099-2106; Patil et al. (2008)
Bioconjug. Chem., 19:1396-1403; Patil et al. (2009)
Biomacromolecules, 10:258-266). It was also demonstrated that
nondegradable PAMAM dendrimers are efficient for siRNA delivery and
induce potent endogenous gene silencing, which was dependent on the
dendrimer generation (Zhou et al. (2006) Chem. Commun. (Camb),
22:2362-2364). On the contrary, it was reported that PAMAM
dendrimers have moderate efficiency for the delivery of
oligonucleotides and are relatively less effective for delivery of
siRNA especially in multidrug resistant cancer cells which
overexpress P-glycoprotein (Kang et al. (2005) Pharm. Res.,
22:2099-2106). Similarly to the PAMAM dendrimers, the gene
silencing ability of the siRNA-PPI dendrimer complex was also
depended on the dendrimer generations and in general siRNA
delivered by the dendrimers with higher generation demonstrated
more efficient gene silencing (Zhou et al. (2006) Chem. Commun.
(Camb), 22:2362-2364). However, it was found that the optimal
generation of PPI dendrimer is G4, which is much lower when
compared with PAMAM dendrimers. It was reported that the best gene
silencing results were obtained with G7 dendrimers at a N/P ratio
of 10-20 (Zhou et al. (2006) Chem. Commun. (Camb), 22:2362-2364),
while only weak gene silencing was observed when PAMAM G5 was used
(Kang et al. (2005) Pharm. Res., 22:2099-2106). PAMAM dendrimers
have relatively larger size compared to PPI dendrimers, which
should be more efficient in complexation of siRNA to form compacted
structures. However, on the other hand, PPI dendrimers contain 100%
protonable nitrogen (van Duijvenbode et al. (1998) Polymer,
39:2657-2664). The existence of multiple amide nitrogen in the
inner structure of the PAMAM, which are nonbasic due to the
delocalization of their lone electron pairs with the carbonyl
group, siRNA binding and proton-sponge capacity of the PAMAM might
be compromised compared to PPI dendrimers.
[0132] It was demonstrated that the molecular structure of a
nanocarrier, including its charge, size, and flexibility coherently
determines siRNA condensation efficiency and the physicochemical
properties of the formed siRNA complexes, which in turn controls
cellular uptake of the siRNA and the silencing efficiency of the
internalized siRNA. PPI dendrimers can be effectively used for
packaging and delivering of siRNA into cancer cells. Quantitative
evaluation of the efficiency of PPI dendrimer to provoke 21-bp
siRNA condensation revealed that all four generations of PPI
dendrimers are capable of siRNA packaging. However, PPI G4 was the
most efficient in siRNA complexation compared to other studied
generations, including PPI G5 dendrimers. Atomic force microscope
studies demonstrated that the lower generations of dendrimers were
capable to partially condensate siRNA to randomly aggregated
nanofibers (G2), or a mixture of nanofibers and thin toroids (G3).
In contrast, the products of siRNA condensation with higher
generations of PPI dendrimers (G4 and G5) appear to be uniform
discrete nanoparticles with average size of 150 nm. Furthermore,
siRNA-PPI G2 complexes did not provide for an efficient
internalization of siRNA by cancer cells. In contrast PPI G3, G4
and G5 dendrimers were capable to facilitate siRNA internalization
into cytoplasm and silence the targeted mRNA. The silencing
efficacy also highly depended on the generations of the PPI
dendrimers, with the following trend: PPI G4>G5>G3>G2.
Example 3
[0133] Design and creation of novel nanometer-size carriers for the
safe delivery of small interfering RNA (siRNA) towards their
potential applications in cancer therapy is one of the challenging
and rapidly growing areas of research. RNA interference (RNAi) is a
conservative biological response to siRNA that regulates the
expression of protein coding genes (Caplen et al. (2003) Ann. N.Y.
Acad. Sci., 1002:56-62; Dave et al. (2003) Rev. Med. Virol.,
13:373-385; Dorsett et al. (2004) Nat. Rev. Drug Discov.,
3:318-329; Mello et al. (2004) Nature 431:338-342; Sontheimer, E.
J. (2005) Nat. Rev. Mol. Cell. Biol., 6:127-138). However, the
broad therapeutic applications of siRNA are limited by major
delivery problems (Paroo et al. (2004) Trends Biotechnol.,
22:390-394). The efficient in vivo gene knock down requires a
delivery system that would overcome the following limitations: (1)
low cellular uptake, (2) poor endosomal escape, (3) substantial
liver and renal clearance, (4) facile enzymatic degradation in the
blood and extracellular environment and (5) inefficient gene
silencing.
[0134] Recent investigations in the area of nanomaterials for RNA
delivery provided solutions to some of the major siRNA delivery
problems (Chen et al. (2009) Small 5:2673-2677; Christie et al.
(2010) Endocrinology 151:466-473; Guo et al. (2010) Adv. Drug
Deliv. Rev., 62:650-666; Ladewig et al. (2010) Biomaterials
31:1821-1829; Minko et al. (2010) Methods Mol. Biol., 624:281-294;
Ozpolat et al. (2010) J. Intern Med., 267:44-53; Patil et al.
(2008) Bioconjug. Chem., 19:1396-1403; Patil et al. (2009)
Biomacromolecules 10:258-266; Schroeder et al. (2010) J. Intern.
Med., 267:9-21; Taratula et al. (2009) J. Control Release
140:284-293; Martin et al. (2007) AAPS J., 9:E18-29). However, the
developed delivery approaches address only selected siRNA delivery
problems lacking optimal balanced delivery system that includes a
solution for all the major aforementioned challenges. For example,
a biodegradable polymer poly-L-lysine (PLL) is being used for gene
delivery and its polyplexes are taken up into cells efficiently.
However, transfection efficiencies of PLL-siRNA complexes remain
several orders of magnitude lower when compared with other
transfection agents. One potential reason for inefficient
transfection has been identified as the lack of amino groups with a
pKa .about.5-7 for so called "proton sponge effect" that offers
endosomolysis and subsequent release of siRNA. The desired
transfection effect was achieved by structural modification of PLL
using a targeting ligand or endosomolytic agents like chloroquine
or fusogenic peptides (Martin et al. (2007) AAPS J., 9:E18-29; Read
et al. (2005) Nucleic Acids Res., 33:e86). A significant
improvement in transfection efficiency was observed when histidine
or imidazole moieties were attached to the PLL (Benns et al. (2000)
Bioconjug. Chem., 11:637-645; Midoux et al. (1999) Bioconjug.
Chem., 10:406-411).
[0135] Another major challenge in the safe delivery of siRNA is its
facile enzymatic degradation in cytoplasm due to the presence of
nucleases that dramatically reduce siRNA half-life. It has been
reported that internally quaternized and cancer-targeted
polyamidoamine (PAMAM) dendrimers provide for the efficient
cellular uptake and excellent gene silencing. It was shown that
surface modification and internal quaternization of dendrimers
reduced their cytotoxicity and substantially improved the cellular
uptake while targeting of the dendrimers to cancer cells initiated
receptor mediated endocytosis and led to the efficient gene knock
down. The importance of free tertiary amine groups in dendrimers
for endosomal escape has also been determined. Here, the design,
synthesis, and evaluation of a triblock delivery system that
provides solutions for major problems in siRNA delivery, i.e. poor
cellular uptake, low endosomal escape, and facile enzymatic
degradation, is provided. A novel triblock nanocarrier
PAMAM-PEG-PLL has been designed to combine individual features of
PAMAM dendrimer, polyethylene glycol (PEG) and poly-L-lysine. PAMAM
dendrimer provides tertiary amines for endosomal escape; PEG covers
up siRNA protecting it from enzymatic degradation; and PLL offers
cationic amine groups for electrostatic interaction with negatively
charged siRNA.
Methods
Materials
[0136] Generation four PAMAM-NH.sub.2 dendrimers (Mw .about.14,214
Da, 64 amine end groups), PLL.HBr (Mw .about.12,000, degree of
polymerization equal to 57), 4-(methylamino)pyridine and methyl
iodide were purchased from Sigma-Aldrich Co. (St. Louis, Mo.).
.alpha., .omega.-Bis(2-carboxyethyl)polyethylene glycol (Mw
.about.3,000 Da) and N-(3-dimethylaminopropyl)-N-ethylcarbodimide
hydrochloride were obtained from Fluka (Allentown, Pa.).
Spectra/Pore dialysis membranes were obtained from Spectrum
Laboratories, Inc. (Rancho Dominguez, Calif.). Ethidium Bromide
(EtBr) solution was purchased from Promega (Madison, Wis.). The
sequence of siRNA targeted to BCL2 mRNA custom synthesized by
Ambion (Austin, Tex.), was: 5'-GUG AAG UCA ACA UGC CUG C-dTdT-3'
(sense strand; SEQ ID NO: 4) and 5'-GCA GGC AUG UUG ACU UCA
C-dTdT-3' (antisense strand; SEQ ID NO: 5). Non-specific siRNA used
as a negative control (sense strand, 5'-CCU CGG GCU GUG CUC UUU
U-dTdT-3', SEQ ID NO: 10; antisense strand, 5'-AAA AGA GCA CAG CCC
GAG G-dTdT-3', SEQ ID NO: 11) was received from Dharmacon Inc.
(Lafayette, Colo.). Fluorescent RNA duplex--siRNA labeled with
Pierce NuLight.TM. DY-547 fluorophores (siGLO Red Transfection
Indicator, red fluorescence) was obtained from Applied Biosystems
(Ambion, Inc., Foster City, Calif.). All other chemicals were
purchased from Fisher Scientific (Fairlawn, N.J.).
Cell Line
[0137] The human ovarian carcinoma A2780 cell line was obtained
from Dr. T. C. Hamilton (Fox Chase Cancer Center). Cells were
cultured in RPMI 1640 medium (Sigma, St. Louis, Mo.) supplemented
with 10% fetal bovine serum (Fisher Scientific, Fairlawn, N.J.).
Cells were grown at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2 (v/v) in air. All experiments were performed on cells in
the exponential growth phase.
Synthesis of Surface Modified PAMAM Dendrimer (PAMAM-NHAc)
[0138] The surface modified and partially acetylated PAMAM-NHAc
dendrimer was prepared as seen in FIG. 14 (Patil et al. (2008)
Bioconjug. Chem., 19:1396-1403). Briefly, triethylamine (0.11 mL,
0.82 mmol) was added to a stirred solution of PAMAM-NH.sub.2
generation four dendrimer (172 mg, 0.012 mmol) dissolved in
anhydrous methanol (10 mL) followed by the addition of excess
acetic anhydride (0.08 mL, 0.72 mmol). The resulting mixture was
stirred at room temperature for 24 hours. Methanol was evaporated
under reduced pressure and the resulting residue was dissolved in
water (2 mL). Further purification by extensive dialysis against
deionizer water using dialysis membrane (molecular mass cut off
2,000 Da) and freeze-drying afforded acetylated PAMAM dendrimer.
The degree of acetylation was confirmed by proton nuclear magnetic
resonance (.sup.1H NMR).
Synthesis of PAMAM-PEG-COOH Conjugate
[0139] .alpha., .omega.-Bis(2-carboxyethyl)polyethylene glycol (15
mg, 5 .mu.mol, Mw.about.3000 Da) and
PAMAM-[(NHAc).sub.58(NH.sub.2).sub.6] dendrimer (83 mg, 5 .mu.mol)
were dissolved in the mixture of anhydrous solvents methylene
chloride (5 mL) and dimethyl sulfoxide (5 mL) (FIG. 14). After
stirring for 10 minutes at room temperature,
N-(3-dimethylaminopropyl)-N-ethylcarbodimide hydrochloride
(EDC.HCl) (1 mg, 5.3 .mu.mol) and 4-(methylamino)pyridine (DMAP)
(0.5 mg) were added to the reaction mixture. The resulting mixture
was stirred for an additional 36 hours at room temperature and
solvents were removed under reduced pressure. The residue was
dissolved in water and purified by extensive dialysis using
Spectra/Por dialysis membrane (molecular weight cutoff, MWC=6,000
Da) against deionized water. The conjugate was further purified by
passing through a sephadex G10 column using water as eluent and
lyophilized to obtain PAMAM-PEG-COOH as a white solid.
Synthesis of PAMAM-PEG-PLL Conjugate
[0140] Triethylamine (0.2 mL) was added to a stirred solution of
poly-L-lysine hydrobromide (22 mg, 1.83 .mu.mole, Mw=.about.12,000,
degree of polymerization equal to 57) in anhydrous dimethyl
sulfoxide (3 mL) (FIG. 14). The reaction mixture was further
diluted with anhydrous methylene chloride (5 mL) followed by the
addition of PAMAM-PEG-COOH conjugate (22 mg, 1.14 .mu.mol) and
stirred at room temperature for 15 min. EDC.HCl (1.5 mg, 7.8
.mu.mol) and DMAP (0.5 mg) were added to the reaction mixture. The
resulting solution was stirred for an additional 36 hours at room
temperature. The side product carbodiimide urea was filtered off
and solvents were removed under reduced pressure. The residue was
dissolved in water and purified by extensive dialysis using
Spectra/Por dialysis membrane (MWC=25,000 Da) against deionized
water. The conjugate was further purified by passing through a
Sephadex.RTM. G10 column using water as eluent and lyophilized to
obtain PAMAM-PEG-PLL as a hygroscopic white solid.
Synthesis of PEG-PLL Conjugate
[0141] NHS-PEG-OMe (15.6 mg, 3.1 .mu.mole, Mw=.about.5000) in
phosphate buffer (pH 8.4) was added to a stirred solution of
poly-L-lysine hydrobromide (22 mg, 1.83 .mu.mole, Mw=.about.12000,
degree of polymerization equal to 57) in phosphate buffer (pH 8.4).
The resulting solution was stirred for 6 hours at room temperature.
The resulting reaction mixture was then dialyzed against 1N HCl for
12 hours and subsequently extensively dialyzed against deionized
water using dialysis membrane Spectra/Por (MWC=8,000 Da). Further
purification by passing through sephadex G10 column using water as
eluent and freeze drying afforded PEG-PLL conjugate.
Synthesis of PAMAM-PEG-PLL-Cy5.5
[0142] Cy 5.5 mono NHS ester (1.5 mg, 1.32 .mu.mol) dissolved in
anhydrous dimethyl sulfoxide (1 mL) was added to a stirred solution
of PAMAM-PEG-PLL (9 mg, 0.32 .mu.mol, Mw .about.28,000) in 0.1 mM
NaHCO.sub.3 (1 mL). The resulting mixture was stirred in the dark
at room temperature for 6 hours. Extensive dialysis using
Spectra/Por dialysis membrane (MWC=25,000) against deionized water
was carried out to remove unreacted Cy 5.5. Additionally, the
conjugate was purified by passing through sephadex column. The
concentration of Cy 5.5 dye attached to the PAMAM-PEG-PLL
nanocarrier was estimated by measuring its fluorescence (Excitation
675 nm, emission 694 nm) using Cy 5.5 NHS ester as standard.
Proton Nuclear Magnetic Resonance Spectroscopy (.sup.1H NMR)
[0143] .sup.1H NMR was performed on a Varian VNMRS 400 MHz NMR
spectrometer (Varian, Inc., Palo Alto, Calif.). The chemical shift
was expressed as parts per million (ppm) and a solvent peak was
used for reference (D.sub.2O, 4.8 ppm). The following abbreviations
are used in the results section to identify multiplicities of
spectra peaks: s, singlet; m, multiplet; br, broad.
In Vitro Cytotoxicity
[0144] A modified MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay was used to assess the cyctotoxicity of the following
nanocarriers PEG, PLL, PEG-PLL, PAMAM-NH.sub.2, PAMAM-OH,
PAMAM-NHAc, and PAMAM-PEG-PLL as previously described (Jayant et
al. (2007) Pharm. Res., 24:2120-2130; Pakunlu et al. (2004) Cancer
Res., 64:6214-6224). To measure cytotoxicity, cells were separately
incubated in a microtiter plate with different concentrations of
PLL, PEG-PLL and PAMAM-PEG-PLL. Control cells received an
equivalent volume of fresh medium. The duration of incubation was
24 hours. On the basis of these measurements, cellular viability
was calculated for each nanocarrier concentration. A decrease in
the cellular viability indicated an increase in the toxicity.
Characterization of siRNA Complexation with Nanocarriers
[0145] The complexes of cationic nanocarriers (PLL, PEG-PLL and
PAMAM-PEG-PLL) and siRNA were prepared in water at N/P
(amine/phosphate) charge ratios ranging from 0 to 1.5 and incubated
at room temperature for 30 minutes. The charge ratio was calculated
by relating the number of cationic primary amine groups on
nanocarrier with the number of negatively charged phosphate groups
of siRNA. Dendrimer-free siRNA was used as the control.
Double-stranded RNA ladder (New England Biolabs, Ipswich, Mass.)
with the smallest base pairs at 21 was used as a size reference.
The samples were further diluted with DPBS buffer and
electrophoresed in 4% agarose gel at 100 V for 50 min in
Tris-Borate-EDTA buffer containing ethidium bromide. siRNA bands on
the gel were visualized under ultraviolet light and photographed.
Complex formation was also quantified by measuring fluorescence of
ethidium bromide in the sample at 530 nm excitation and 590 nm
emission wavelengths (Bolcato-Bellemin et al. (2007) Proc. Natl.
Acad. Sci., 104:16050-5). The fluorescence intensity at N/P charge
ratio equal to 0 was set to 100%.
Dynamic Light Scattering (DLS) Analysis and Zeta Potential
[0146] PAMAM-PEG-PLL-siRNA complex was prepared by mixing
PAMAM-PEG-PLL and siRNA in water at N/P ratio equal to 3. The
resulting complex was incubated for 30 minutes and the size was
determined using the DynaPro-MS800 dynamic light
scattering/molecular sizing instrument with argon laser wavelength
.lamda.=830 nm, a detector angle 90.degree., and typical sample
volume of 20 .mu.L. Each light scattering experiment consisted of
20 or more independent readings, 10 seconds in duration each. Data
analysis was conducted using DynaPro Instrument Control Software
for molecular Research DYNAMICS (version 5.26.60). The obtained DLS
data represents the average of three runs. Zeta potential was
measured on PALS Zeta Potential Analyzer (Brookhaven Instruments
Corp, New York, N.Y.). Samples were taken as is and their volume
was 1.5 mL. All measurements were carried out at room temperature.
Each parameter was measured 5 times, and average values were
calculated.
Cellular Internalization
[0147] Cellular uptake and intracellular localization of siRNA was
investigated using a confocal microscopy. In this experiment,
living cancer cells were incubated with fluorophore labeled naked
siRNA (siGLO.RTM. Red, red fluorescence) and siRNA complexed with
cationic nanocarrier PAMAM-PEG-PLL (N/P=3). Cellular uptake studied
substances was monitored in living cells placed in a chamber at
37.degree. C. within 1 hour. Cellular localization of siRNA was
examined on fixed and washed cells after the incubation for 24
hours with the substances. Fluorescence and its distribution within
the cell were examined using a confocal microscope.
Gene Expression
[0148] Reverse transcription-polymerase chain reaction (RT-PCR) was
used for the analysis of gene expression (Pakunlu et al. (2004)
Cancer Res., 64:6214-6224). The cationic nanonarriers (PLL,
PLL-PEG-OMe and PAMAM-PEG-PLL)-siRNA (for BCL2 gene) complexes were
added to the cells with the final concentration of siRNA equal to 1
.mu.M. After 24 hours, total cellular RNA was isolated using an
RNeasy kit (Qiagen, Valencia, Calif.). First-strand cDNA was
synthesized by Ready-To-Go.TM. You-Prime First-Strand Beads (GE
Healthcare, Piscataway, N.J.) with 2 .mu.g of total cellular RNA
and 100 ng of random hexadeoxynucleotide primer (Amersham
Biosciences, Piscataway, N.J.). After synthesis, the reaction
mixture was immediately subjected to polymerase chain reaction
(PCR), which was carried out using GenAmp PCR System 2400
(Perkin-Elmer, Shelton, Conn.). .beta.2-microglobulin (.beta.2-m)
was used as an internal standard. The following pairs of primers
were used: BCL2: 5'-GGA TTG TGG CCT TCT TTG AG-3' (sense; SEQ ID
NO: 6), 5'-CCA AAC TGA GCA GAG TCT TC-3' (antisense; SEQ ID NO: 7);
.beta.2-m (internal standard)-ACC CCC ACT GAA AAA GAT GA (sense;
SEQ ID NO: 8), ATC TTC AAA CCT CCA TGA TG (antisense; SEQ ID NO:
9). PCR regimen was as follows: 94.degree. C. for 5 minutes;
94.degree. C. for 1 minute, 55.degree. C. for 1 minute, and
72.degree. C. for 1 minute for 41 cycles; and 60.degree. C. for 10
minutes. PCR products were separated in 4% NuSieve 3:1
Reliant-agarose gels (Lonza, Basel, Switzerland) in 1.times.
Tris-borate EDTA buffer [0.089 mol/L Tris-borate, 0.002 mol/L EDTA
(pH 8.3); Research Organics Inc., Cleveland, Ohio] by submarine
electrophoresis. The gels were stained with EtBr and digitally
photographed.
Serum Stability of siRNA and PAMAM-PEG-PLL-siRNA Complex
[0149] Serum stabilities of naked siRNA and siRNA complexed with
PAMAM-PEG-PLL nanocarrier were investigated by incubating siRNA or
PAMAM-PEG-PLL-siRNA complex in 50% human serum at 37.degree. C. Ten
samples were prepared by mixing siRNA (30 nmol) in water with
PAMAM-PEG-PLL (74.8 nmol, N/P=3) solution in water and separately
incubated for 30 minutes at room temperature. In the case of naked
siRNA, equal volume of RNAase free water was used instead of
nanocarrier. To each of these samples 50% human plasma was added
(final siRNA concentration was 1.43 nM) and incubated at 37.degree.
C. Samples were removed at an indicated time intervals (0, 0.25,
0.5, 0.75, 1, 3, 6, 12, 24 and 50 hours) and analyzed using a gel
electrophoresis (4% agarose gel at 100 V for 50 minutes) in
Tris-Borate-EDTA buffer containing EtBr. siRNA bands on the gel
were visualized under ultraviolet light. PAMAM-PEG-PLL-siRNA
samples were pre-treated with polymethacrylic acid to release free
siRNA from cationic nanocarriers. 100 .mu.L of polymethacrylic acid
solution (4 .mu.M) was added to the complexes (triblock
nanocarrier/siRNA=3) and incubated at 37.degree. C. with 50% human
plasma. The released siRNA was then analyzed by a gel
electrophoresis.
Statistical Analysis
[0150] Data obtained were analyzed using descriptive statistics,
single factor analysis of variance (ANOVA) and presented as a mean
value.+-.standard deviation (SD) from five independent
measurements. Data sets were analyzed for significance with
Student's t test and considered P values of less than 0.05 as
statistically significant.
Results
[0151] Delivery of siRNA into the cytoplasm of cancerous cells
where it triggers sequence specific mRNA degradation has recently
emerged as a powerful tool in the gene therapy. The major obstacles
in safe transportation of siRNA have been extensively investigated
and condensation to nanoparticles has now been recognized as the
most efficient method for facile transport of siRNA. Therefore,
designing a nanocarrier that enables effective and safe transfer of
siRNA into mammalian cells is a task of great interest. In general,
combining multiple functions in a single delivery system is a
difficult task and requires laborious synthetic efforts. Herein,
the design, synthesis, and evaluation of a synthetically simple yet
novel triblock multifunctional nanocarrier PAMAM-PEG-PLL that
effectively combines three functionalities which are otherwise
ineffective when tested individually is described. The triblock
nanonarrier PAMAM-PEG-PLL serves three distinct functions: (1) PLL
provides cationic primary amine groups for electrostatic
interaction with negatively charged siRNA (2) PAMAM dendrimer
offers necessary tertiary amine groups for proton sponge effect,
while (3) PEG confers nuclease stability in blood serum.
[0152] A three step synthetic route was used for the preparation of
PAMAM-PEG-PLL nanocarrier (FIG. 14). In the first step, PAMAM
dendrimer was partially acetylated to afford
PAMAM-[(NHAc).sub.58(NH.sub.2).sub.6] dendrimer. The second step
involved synthesis of PAMAM-PEG-COOH by reacting one of the acid
group of .alpha., .omega.-bis(2-carboxyethyl)polyethylene glycol
(Mw=.about.3000) with one of the primary amine of
PAMAM-[(NHAc).sub.58(NH2).sub.6] dendrimer. One could expect that
the length of the PEG block would have great influence on the final
triblock-siRNA complex as a shorter length might not be enough to
protect siRNA from the enzymatic degradation. Instead of using a
single long PEG chain length a medium size chain (MW 3000) was
used, while the ratio of triblock nanocarrier/siRNA was taken as 3
(N/P=3). Thus, a single siRNA is surrounded by 3 PEG blocks and
expected to give effective protection. Previously, the cellular
penetration of PEG with different molecular weight (up to MW 20000
Da) was tested and it was found that PEG with MW around 3000 Da
penetrate cells faster when compared with higher MW polymers
(Chandna et al. (2007) Mol. Pharm., 4:668-78). Based on these
considerations, PEG with MW 3000 Da was selected. During the third
step, terminal free acid group of PAMAM-PEG-COOH was reacted with
PLL using EDC as a coupling reagent.
[0153] One can assume that the synthetic procedure used could not
be selective and the design led to some cross-linking reactions.
However, the instant synthetic scheme can be selective because it
is a sequential procedure and the following reasons contribute to
the selective formation of PAMAM-PEG and not a PAMAM-PEG-PAMAM
cross-linking polymer: (1) PAMAM-NHAc has only 5-6 free amines to
react with the PEG-dicarboxylic acid; (2) the free amine groups on
dendrimer are crowded with NHAc substituent and not easily
available for cross-linking; (3) dilution of reaction also plays an
important role in reducing the cross-linking reaction; (4) this
reaction greatly differs from the usual small molecules where
selectivity would be an issue; (5) in the present studies, more
harsh conditions, prolonged reaction time, and more equivalents of
coupling reagents would be required to force the cross-linking
PAMAM-PEG-PAMAM reaction; (6) the technique of filtering the
reaction mixture after coupling reaction was followed to remove any
insoluble material, however, in this reaction, negligible amount of
insoluble material was formed; (7) even if the cross-linking
product is formed, it was in trace amount and removed completely
during filtration. The coupling reaction of PAMAM-PEG with PLL can
be selective to form the desired PAMAM-PEG-PLL as PLL has several
amine groups available to react in comparison with 5-6 amine groups
of PAMAM dendrimer. The .sup.1H-NMR was very useful and informative
in determining the content of nanocarrier. The area under peak
would greatly vary (almost double or half) if the number of units
(PAMAM, PEG and PLL) changes in the final nanocarrier. Each block
has a large number of protons and cannot be ignored.
[0154] Theoretically, there might be a possibility that some
molecules of siRNA could form complexes directly with the PAMAM
dendrimer, not with positively charged PLL. The PAMAM dendrimer
used in present study was surface modified and lacks free primary
amine groups required to form complex with siRNA. As .sup.1H-NMR
studies show, very few (.about.5-6) free amines are available on
the PAMAM dendrimer. Such an amount is not enough to form complexes
with siRNA that possess about 42 negatively charged phosphate
groups. It has been suggested that tertiary amines in PAMAM
dendrimer do not participate in direct complexation with siRNA and
stable complex is formed only after quaternizing the internal
tertiary amine groups (Patil et al. (2009) Biomacromolecules
10:258-266). This supports the fact that PAMAM dendrimer would not
form complex with siRNA.
[0155] The following three matrix compounds were used to record
MALDI-TOF: sinapinic acid, .alpha.-cyano-4-hydroxycinnamic acid,
and 2,5-dihydroxybenzoic acid. More than likely, the triblock
nanocarrier did not ionize under the attempted conditions. All
proton NMR spectra were recorded in solution of studied compounds
in D.sub.2O using a 400 MHz NMR spectrometer. The chemical shift
(.delta.) was expressed as parts per million (ppm). The data
obtained confirmed the structures of synthesized substances.
PAMAM-NHAc .sup.1H NMR spectral data are shown in FIG. 15A. The
following peaks were identified: .delta. 1.98 (s, COCH.sub.3),
2.40-2.50 (br m, CH.sub.2CONH), 2.62-2.73 (br m,
CONHCH.sub.2CH.sub.2N), 2.82-2.92 (br m, NCH.sub.2CH.sub.2CONH),
3.26-3.37 (m, CONHCH.sub.2 and CH.sub.2NHCOCH.sub.3). The degree of
acetylation (.about.90%) was confirmed from the proton NMR spectra
(FIG. 15A) by calculation the ration between the integrated peak
area of signal appeared at .delta. 1.98 ppm (--NHCOCH.sub.3) to
that of methylene protons of PAMAM dendrimer (.delta. 2.40-3.37).
PAMAM-PEG-COOH .sup.1H NMR spectrum (FIG. 15B) showed the following
peaks: .delta. 2.00 (s, COCH.sub.3), 2.40-2.50 (br m,
CH.sub.2CONH), 2.64-2.75 (br m, CONHCH.sub.2CH.sub.2N), 2.82-2.92
(br m, NCH.sub.2CH.sub.2CONH), 3.30-3.38 (m, CONHCH.sub.2 and
CH.sub.2NHCOCH.sub.3), 3.74 (s, --CH.sub.2CH.sub.2O). The spectra
showed mono acylation of dendrimer with .alpha.,
.omega.-bis(2-carboxyethyl)polyethylene glycol leaving another
--COOH group free for conjugation with poly-L-lysine (PLL). The
.sup.1H-NMR spectra for this compound confirmed the presence of
both polyethylene glycol and dendrimer protons. Further mono
functionalization was determined by calculating the area under
proton peaks arising from polyethylene glycol (Mw=-3000,
--CH.sub.2CH.sub.2O--, .about.270H) and dendrimer (--COCH.sub.3,
174H) appeared at .delta. 3.74 and .delta. 2.00, respectively.
PAMAM-PEG-PLL .sup.1H-NMR spectral data are shown in FIG. 15C. The
following peaks were identified: .delta. 1.30-1.50 (br m, PLL),
1.60-1.90 (br m, PLL), 1.98 (s, PAMAM), 2.40-2.50 (br m, PAMAM),
2.60-2.70 (br m, PAMAM), 2.78-2.88 (br m, PAMAM), 2.90-3.05 (br m,
PLL), 3.26-3.37 (m, PAMAM), 3.70 (s, PEG), 4.25-4.35 (br m, PLL).
The spectra confirmed the formation of PAMAM-PEG-PLL nanocarrier
showing the presence of proton peaks arising from dendrimer
(PAMAM), polyethylene glycol (PEG) and poly-L-lysine (PLL). PEG-PLL
.sup.1H-NMR spectrum (FIG. 15D) showed the following peaks: .delta.
1.38-1.60 (br m, --CH.sub.2--CH.sub.2--CH.sub.2--), 1.68-1.90 (br
m, --CH.sub.2--CH.sub.2--CH(CO)NH-- &
--CH.sub.2--CH.sub.2--CH.sub.2NH.sub.2--), 2.90-3.10 (br m,
--CH.sub.2--CH.sub.2NH.sub.2--), 3.70 (s, --CH.sub.2CH.sub.2O PEG),
4.30-4.40 (br m, --CH.sub.2--CH.sub.2--CH(CO)NH--). .sup.1H-NMR
technique has been widely used and documented as one of the best
method for calculating the functional group content of dendrimers.
The number of amine groups left free after acetylation reaction can
be calculated using the .sup.1H-NMR technique (Majoros et al.
(2003) Macromolecules 36:5526-5529). Since the triblock nanocarrier
is a combination of linear as well as spherical polymer, it will
not follow the behavior of the conventional polymer molecules and
therefore, the conventional molecular weight determination method
based on calibration cannot be used in such cases. More studies and
special set-ups are required to use the Gel Permeation
Chromatography (GPC) method to determine the molecular weight. It
was found that .sup.1H-NMR spectroscopy was highly useful and
provided the desired information by comparing the area under peak.
The number of free amines in the PAMAM-PEG-PLL nanocarrier was
calculated based on the degree of polymerization in the
poly-L-lysine polymer. There could be a very small variation in the
calculated and actual content of functional group in the
nanocarrier however it would not greatly affect its function.
Gel-electrophoresis studies also provided indirect evidence on
accuracy of calculated content of functional group in triblock
nanocarriers. The band for siRNA was completely disappeared when
the ratio of N/P was 1, i.e., the number of cationic
amines/phosphate of siRNA is 1/1. If the calculated amount of
cationic amines is not correct the nanocarrier/siRNA complex
formation can be expected at different N/P ratio (--NH.sub.2 of
nanocarrier/phosphate of siRNA).
[0156] The measurement of viability of cells incubated with
different concentrations of PEG, PLL, PEG-PLL, PAMAM-NHAc-PEG and
PAMAM-NHAc-PEG-PLL compounds showed their relatively low
cytotoxicity (FIG. 16A). No substantial differences were found
between different nanocarriers under the concentrations of the
compounds lower than 4 .mu.M. However, the concentrations exceed 4
.mu.M, PEG-PLL demonstrated higher cytotoxicity when compared with
PLL alone and PAMAM-NHAc-PEG-PLL. Therefore, the toxicity of
PEG-PLL was reduced when PEG-PLL was conjugated to PAMAM-NHAc
dendrimer. Cytotoxicity data for the triblock PAMAM-NHAc-PEG-PLL
nanocarrier were compared with cytotoxicity of its previously
synthesized predecessors (PAMAM-NH.sub.2, PAMAM-OH, and PAMAM-NHAc
dendrimers) (Patil et al. (2008) Bioconjug. Chem., 19:1396-1403).
The results of such comparison showed a comparably low cytotoxicity
for triblock PAMAM-NHAc-PEG-PLL and acetylated PAMAM-NHAc
dendrimers. The cytotoxicity under the high concentrations of both
dendrimers was lower when compared with quaternized non-acetylated
PAMAM-OH dendrimer. In contrast, non-modified PAMAM-NH.sub.2
dendrimer demonstrated a significant cellular toxicity under the
concentrations higher than 5 .mu.M. It should be stressed that a
maximum decrease in viability of cells incubated with PEG, PLL,
PEG-PLL, PAMAM-NHAc-PEG-PLL, PAMAM-OH, and PAMAM-NHAc compounds was
substantially higher than 50% under all studied concentrations.
Such low toxicity does not allow calculating the IC.sub.50 dose for
these substances (half maximal inhibitory concentration, the dose
that kills about 50% of cells). In contrast, the IC50 dose of
non-modified PAMAM-NH.sub.2 dendrimer was estimated to be around 6
.mu.M (FIG. 16A).
[0157] The nanocarrier-siRNA complex formation and optimal N/P
ratio was determined by agarose gel electrophoresis. The PLL,
PEG-PLL and PAMAM-PEG-PLL nanocarriers were mixed with siRNA in
water at various N/P charge ratios and were subjected to
electrophoresis in agarose gel (FIG. 16B). The numbers of cationic
primary amine groups in PLL, PEG-PLL and PAMAM-PEG-PLL were
calculated based on PLL Mw (.about.8,000) and degree of
polymerization (57). All three nanocarriers showed the complex
formation at N/P ratio 1 and above as evidenced by oligonucleotide
bands disappearance from agarose gels. The quantitative analysis
showed that fluorescence of PLL-siRNA, PEG-PLL-siRNA and
PAMAM-PEG-PLL-siRNA progressively decreased with the increase in
N/P ratio. For PLL-siRNA, the complexation decreased fluorescence
to 19%, 8% and 0% with N/P ratio equal to 0.5, 1.0, 1.5 relative
units, respectively; for PEG-PLL-siRNA, fluorescence decreased to
19%, 8%, 0% with N/P ratio equal to 0.5, 1.0, 1.5 relative units,
respectively; for PAMAM-PEG-PLL-siRNA, fluorescence decreased to
45%, 0% with N/P ratio equal to 0.5, 1.0, respectively.
[0158] The hydrodynamic diameter of PAMAM-PEG-PLL-siRNA complex was
determined by dynamic light scattering at charge ratio ranging from
1 to 3 rel. units. The PAMAM-PEG-PLL/siRNA particle size slightly
decreased with increasing the charge ratio to 3 relative units
(FIG. 16C). The measurements of zeta potential of dendrimer-siRNA
complexes showed that the complexes were neutral. The size of
PAMAM-PEG-PLL-siRNA complexes used in this paper varied from 120 to
180 nm depending on N/P ratio. This relatively large size is
attributed to cross-linking of nanocarrier and siRNA. It has been
shown that an internally charged dendrimer gives small, compact,
and spherical nano-particles with siRNA. However, commercial
PAMAM-NH.sub.2 showed nano-fibers due to cross-linking (Chandna et
al. (2007) Mol. Pharm., 4:668-78). In the present experiments, a
similar trend is expected because PLL possess primary amine
groups.
[0159] The cellular uptake of naked and complexated fluorophore
labeled siRNA (siGLO.RTM. Red, red fluorescence) was studied in
living (not washed and fixed) cells using confocal microscopy.
A2780 human ovarian cancer cells were incubated with free siRNA and
PAMAM-PEG-PLL-siRNA complex, and were subjected to confocal
microscopy. Naked siRNA did not penetrate the cancer cells (FIG.
17A). It has been reported that PAMAM-NH.sub.2 and PAMAM-OH
dendrimers failed to deliver siRNA into cells, while the
acetylation of PAMAM dendrimer surface substantially improved
internalization of PAMAM-siRNA complexes (Patil et al. (2008)
Bioconjug. Chem., 19:1396-1403). Based on this finding, a PAMAM
dendrimer with the acetylated surface further modified with PEG and
PLL was used. It was found that siRNA complexated with a
PAMAM-PEG-PLL cationic nanocarrier provided excellent cellular
uptake (FIG. 17B). Moreover, optical sections z-series of a single
living cell showed the homogenous and uniform distribution of
siRNA-dendrimer complex in different cellular layers from the top
of cell to the bottom (FIG. 17C). All experiments were performed on
living cells without staining, fixation, and washing out the media
with fluorescently labeled siRNA-dendrimer complexes. Because of
this one can see red fluorescence both inside the cells and media.
In contrast naked siRNA could be seen just in the media but not in
the cell (FIG. 17A). The stability of siRNA in the blood serum was
determined by incubating siRNA either naked or complexed with
PAMAM-PEG-PLL nanocarrier in the human blood serum (FIG. 18). As
expected, naked siRNA started to degrade after 1 hour of incubation
and completely degraded within 12 hours. In contrast, complexation
of siRNA to PAMAM-PEG-PLL nanocarrier protected siRNA from the
nuclease degradation; even 48 hours after the incubation of
complexated siRNA with human blood serum, siRNA remained
nondegraded. Therefore the proposed complexation of siRNA with
PAMAM-PEG-PLL prevents the degradation of siRNA in the plasma.
[0160] The gene knockdown efficiency of siRNA delivered by
Poly-L-Lysine (PLL), PEG-PLL, PAMAM, PAMAM-PEG and PAMAM-PEG-PLL
nanocarriers with appropriate controls (fresh media, naked specific
siRNA, naked non-specific siRNA with scrambled sequence and
non-specific siRNA delivered by PAMAM-PEG-PLL nanocarrier) was
investigated using quantitative RT-PCR. BCL2 protein responsible
for cellular antiapoptotic defense was selected as a target for
siRNA. The results of these experiments are shown in FIG. 19. It
was found that siRNA delivered by PAMAM, PAMAM-PEG, PLL, and
PEG-PLL nanocarriers lowered the expression of the targeted gene
approximately up to 70-50% from its control value (FIG. 19, bars 2,
3, P<0.05). In contrast, delivery of siRNA by a PAMAM-PEG-PLL
triblock nanocarrier led to a significant suppression of the
expression of the targeted BCL2 gene down to 20% from the control
value (P<0.05). The decrease in gene expression after incubation
with PAMAM-PEG-PLL-siRNA was statistically significant (P<0.05)
when compared with either PLL-siRNA or PEG-PLL-siRNA complexes. It
should be stressed that naked BCL2-specific siRNA, naked
non-specific siRNA and non-specific BCL2 siRNA conjugated with
PAMAM-PEG-PLL did not influenced on the expression of BCL2
mRNA.
[0161] Studies on PLL as a cationic nanocarrier for gene
transfection efficiency revealed that PLL alone provides relatively
low gene knockdown, which is attributed to the lack of tertiary
amine groups for the so called proton sponge effect. It is believed
that this effect plays a substantial role in endosomal escape of
siRNA inside cells after endocytosis (Inoue et al. (2008) J.
Control Release 126:59-66; Kano et al. (2011) J. Control Release
149:2-7; Nel et al. (2009) Nat. Mater., 8:543-557). Nevertheless,
PLL in combination with proton sponge ligands such as imidazole or
histidine effectively reduced the gene expression. However, the
toxicity of PLL dramatically decreased when imidazole or histidines
were attached to PLL (Benns et al. (2000) Bioconjug. Chem.,
11:637-645; Midoux et al. (1999) Bioconjug. Chem., 10:406-411). In
the present investigation, a combination of PLL with a nontoxic
PAMAM-NHAc dendrimer that possess several internal tertiary amine
groups is provided. These groups will induce osmotic swelling of
the endosome due to endosomal buffering and lead to the rupture of
endocytotic vesicles and subsequent release of their payload.
Furthermore, polyethylene glycol (PEG) was included in the
nanocarrier to enhance siRNA stability against nuclease enzymes
during the voyage in the human blood stream. A decrease in
cytotoxicity of PLL by attaching a nontoxic PAMAM-NHAc dendrimer
and polyethylene glycol was achieved.
[0162] The ability of PLL, PEG-PLL and PAMAM-PEG-PLL to form
complex with siRNA was compared using agarose gel electrophoresis
method. All three nanocarriers formed a stable complex at N/P ratio
1 and above. The numbers of cationic primary amine groups were
calculated based on PLL molecular weight and degree of
polymerization (.about.8000 Da and 57, respectively). Each
PAMAM-PEG-PLL carrier (calculated Mw 27650 Da) contained
approximately 56 primary amine groups. Similarly, cationic groups
for PEG-PLL (calculated Mw 11,000 Da, DP 57) and PLL (Mw 8000 Da,
DP 57) were calculated as 56 and 57 respectively. As expected, the
agarose gel electrophoresis data showed that PAMAM-PEG-PLL showed
similar to PEG-PLL and PLL ability to form complexes with siRNA.
Dynamic light scattering data revealed an average size around 150
nm of the resulting complexes of the proposed nanocarriers with
siRNA. This size of the resulting nanoparticles and possible impact
of PLL as penetration enhancer resulted in the efficient cellular
uptake of triblock nanocarrier PAMAM-PEG-PLL-siRNA complexes by
human cancer cells.
[0163] However, effective uptake of siRNA by cells does not
automatically ensure effective silencing of its targeted mRNA. For
instance, previously, it has been shown that an effective
intracellular delivery of siRNA by dendrimers does not guarantee
its high gene silencing activity (Patil et al. (2008) Bioconjug.
Chem., 19:1396-1403; Patil et al. (2009) Biomacromolecules
10:258-266). Down regulation of specific gene by siRNA can be
controlled by two possible contributing factors (1) effective
cellular internalization of siRNA and (2) endosomal escape of the
payload to perform the task. Some cationic polymers used for siRNA
delivery including PLL polymer show an excellent penetration into
the cells, while demonstrating a relatively weak gene knockdown due
to poor endosomal release of the siRNA payload (Hwang et al. (2001)
Curr. Opin. Mol. Ther., 3:183-191). The PAMAM dendrimer unit in the
triblock of the proposed nanocarrier PAMAM-PEG-PLL provides the
required tertiary amines for proton sponge effect and subsequent
endosomal release of the siRNA. The proton sponge effect is only
one possible mechanism of the release of siRNA from the complex.
The following mechanisms can potentially be involved in the
intracellular release of siRNA. First, siRNA-carrier complex enters
the cells by endocytosis in membrane limited endosomes that
eventually fuse with lysosomes. This leads to the sharp decrease in
pH disrupting electrostatic interactions between the nucleic acid
and carrier and ultimately leading to the siRNA release. Secondly,
lysosomal enzymes and the acidic environment can either degrade or
swell polymers stimulating the release siRNA from the nanoparticle
(Gary et al. (2007) J. Control Release 121:64-73). Thirdly,
polymers can themselves possess some membrane disruptive
properties. They can swell and burst the endosome through
protonation of excess amine groups (Putnam et al. (2001) Proc.
Natl. Acad. Sci., 98:1200-5).
[0164] Thus, PAMAM-PEG-PLL nanocarrier fulfills both the
requirements of an effective delivery system of improved
penetration and delivery of siRNA to the cytoplasm to achieve
desired gene knockdown. The role of the PAMAM dendrimer was
confirmed by comparing the gene silencing efficiency of BCL2 gene
of siRNA complexed with triblock PAMAM-PEG-PLL, PLL and PEG-PLL
nanocarriers. Indeed the triblock nanocarrier PAMAM-PEG-PLL-siRNA
showed maximum suppression of the expression of targeted BCL2 gene
while PLL alone or in combination with poly(enthylene glycol)
(PLL-PEG) led to a substantially lower decrease in the expression
of this gene.
[0165] After confirming the role of PLL and PAMAM in the triblock
nanocarrier PAMAM-PEG-PLL, the role of PEG to protect the siRNA
during the voyage in the human blood stream was examined. Nuclease
enzyme degradation of siRNA in the blood serum is one of the major
obstacles for the in vivo therapeutic applications of the siRNA.
PEGylation of siRNA or nanocarriers greatly improved the stability
of the siRNA in the human blood serum (Kim et al. (2006) J. Control
Release 116:123-129; Merkel et al. (2009) J. Control Release
138:148-159; Sato et al. (2007) J. Control Release 122:209-216;
Schiffelers et al. (2004) Nucleic Acids Res., 32:e149; Taratula et
al. (2009) J. Control Release 140:284-93). Though the exact
mechanisms of such stabilization are not clear, one can assume that
siRNA is shielded by a linear polymer polyethylene glycol and thus
minimizes its exposure to the nuclease enzymes. This assumption is
based on the following considerations. Although, PEG is a middle
block of the nanocarrier, it is also a hydrophilic segment and
therefore one can expect a micelle like geometry of the complex.
The triblock nanocarrier on complexation with siRNA may form
micelle wherein the hydrophilic region (PEG) encapsulates PLL/siRNA
complex. As expected, siRNA complexed with the proposed triblock
nanocarrier PAMAM-PEG-PLL showed excellent siRNA stability in human
blood serum. In fact, complexated siRNA was stable in the human
serum more than 48 hours, while naked siRNA degraded in less than 6
hours.
[0166] A triblock nanocarrier was designed, synthesized, and
evaluated for the efficient delivery of siRNA. The multifunctional
triblock nanocarrier is synthetically simple to prepare and provide
a solution to several obstacles involved in therapeutic
applications of siRNA.
Example 4
[0167] CD44 (Cluster of Differentiation 44) is a type I
transmembrane protein and a member of the cartilage link protein
family. It is involved in cell-cell and cell-matrix interactions
and signal transduction. CD44 is one of the major determinants of
multidrug resistance and metastases in many types of cancers. CD44
binds to hyaluronic acid found in all types of extracellular
matrices and is a major constituent of the peritoneum, a common
site for ovarian cancer metastases. It has been found that this
protein is overexpressed in several types of gynecological cancers,
especially in tumor tissues of patients with ovarian carcinoma
(FIG. 20). Moreover its expression in tumor tissue statistically
significantly exceeds the expression in normal tissue of the same
organ and from the same patient. Consequently, CD44 protein may be
suppressed in cancer cells in order to enhance the efficacy of
chemotherapy of primary cancer and prevent the development of
metastases.
[0168] Naked siRNA targeted to CD44 and scrambled siRNA were not
efficient in such suppression (FIG. 21). However, anti-CD44 siRNA
delivered with a special carrier--PPI dendrimer led to the
suppression the expression of CD44 protein in cancer cells isolated
from ascetic fluid taken from patients with metastatic ovarian
cancer. Targeting of PPI dendrimer to cancer cells by LHRH peptide
substantially enhanced the suppression of this protein by the
delivered siRNA. The gene expression data obtained using
quantitative reverse transcription PCR (FIG. 21) were confirmed by
registration of protein expression using fluorescence microscopy
(FIG. 22). In the later experiments, CD44 proteins were labeled
with anti-CD44 antibody (red fluorescence) while cellular nuclei
were labeled by nuclear stain (blue fluorescence). In non treated
cancer cells, CD44 proteins are overexpressed predominately in the
plasma membrane. Treatment of cancer cells with LHRH
peptide-targeted PPI dendrimer containing anti-CD44 siRNA
substantially decreased the expression of CD44 in the plasma
membrane of cancer cells. Measurement of cytotoxicity of paclitaxel
(FIG. 23) showed that the delivery of this drug by PPI dendrimer
and simultaneous suppression of CD44 protein substantially enhanced
cytotoxicity of the drug.
Example 5
[0169] The ability of short interfering RNA (siRNA) to silence
specific genes inspired the use of siRNA as a therapeutic agent for
a wide spectrum of disorders including cancer, infectious diseases,
and metabolic disturbances (Devi, G. R. (2006) Cancer Gene Therapy
13:819-829; Garbuzenko et al. (2009) Pharmaceutical Res.,
26:382-394; Chang et al. (2006) Gene Therapy 13:871-872; Rozema et
al. (2007) Proc. Natl. Acad. Sci., 104:12982-12987; Betigeri et al.
(2006) Mol. Pharm., 3:424-430). The main advantages of RNA
interference compared to other therapeutic approaches include
exceptional specificity of siRNA, high potency of gene silencing,
and the ability to target virtually any expressed gene (Dykxhoorn
et al. (2005) Ann. Rev. Med., 56:401-423; Uprichard, S. L. (2005)
FEBS Lett., 579:5996-6007). However, the low penetration ability of
naked siRNA into the cellular cytoplasm to induce sequence-specific
mRNA degradation represents a primary obstacle limiting the success
of siRNA therapy (Uprichard, S. L. (2005) FEBS Lett.,
579:5996-6007; Gary et al. (2007) J. Controlled Release 121:64-73;
Ikeda et al. (2006) Pharm. Res., 23:1631-1640; Akhtar et al. (2007)
J. Clin. Invest., 117:3623-3632; Crombez et al. (2007) Biochem.
Soc. Trans., 35:44-46). Despite extensive research, an efficient,
nontoxic gene delivery approach has not yet been developed. It is
recognized that the delivery of the nucleic acid by nanocarriers
facilitates the cellular uptake of DNA/siRNA and increases their
gene silencing ability (Medarova et al. (2007) Nat. Med.,
13:372-377; Patil et al. (2008) Bioconjugate Chem., 19:1396-1403;
Saad et al. (2008) Nanomed., 3:761-776). Viruses have been studied
as gene delivery vectors; however, the immune response elicited by
viral capsid proteins represents a major challenge limiting the
wide use of this approach (Bessis et al. (2004) Gene Ther.,
11:S10-S17). Consequently, considerable interest to the development
of nonviral gene delivery vehicles has been generated. In order to
provide effective gene silencing, two controversial requirements
for such delivery systems should be satisfied: (1) stability of
siRNA carrier complex during its journey in the systemic
circulation toward the targeted cells and the protection of the
payload against the aggressive biological environment and (2)
intracellular availability of the nucleic acids in order to permit
desired therapeutic effects within the cells (Gary et al. (2007) J.
Controlled Release 121:64-73; Ogris et al. (1999) Gene Ther.,
6:595-605; Oupicky et al. (2001) Gene Ther., 8:713-724; Taratula et
al. (2009) J. Controlled Release 140:284-293).
[0170] In order to optimize the delivery of siRNA and enhance the
efficiency of the treatment, it is highly desirable to employ
clinically relevant imaging approaches for in-situ monitoring of
the disease progression and therapeutic responses (Medarova et al.
(2007) Nat. Med., 13:372-377). Magnetic Resonance Imaging (MRI) is
a powerful tool for non-invasive in vivo monitoring due to its high
resolution and lack of ionizing radiation (Wang et al. (2008) CA
Cancer J. Clin., 58:97-110; Atri, M. (2006) J. Clin. Oncol.,
24:3299-3308). Superparamagnetic Iron Oxide (SPIO) nanoparticles
have been widely investigated as MRI contrast agents to enhance
images of biological molecules (Thorek et al. (2006) Ann. Biomed.
Engin., 34:23-38; Lee et al. (2009) Angew. Chemie-Intl. Ed.,
48:4174-4179). Moreover, several approaches have been reported for
both siRNA and DNA delivery based on SPIO nanoparticles to timely
monitor the delivery process and also to evaluate the therapeutic
effects (Medarova et al. (2007) Nat. Med., 13:372-377; Boyer et al.
(2010) J. Mater. Chem., 20:255-265; Pan et al. (2007) Cancer Res.,
67:8156-8163). However, these methods have various shortcomings and
do not allow a balanced optimization of siRNA compaction, endosomal
escape, and dissociation from the nanoparticles. For example,
covalently linked siRNA molecules to the SPIO surface and
demonstrated the feasibility of using SPIO nanoparticles as MRI
enhancers for in vivo tracking of tumor uptake and silencing
effects of the siRNA (Medarova et al. (2007) Nat. Med.,
13:372-377). However, siRNA molecules in this study are tethered to
the nanoparticles through chemical bonds between the siRNA and SPIO
nanoparticles. Consequently, it is highly possible that such
chemical conjugations might potentially compromise the silencing
effects of siRNA. Moreover, a chemical conjugation might also limit
the siRNA loading capacity of the SPIO nanoparticles. In addition,
cellular uptake of existing SPIO-siRNA complexes is not limited
only to the targeted cells. Consequently, such non-targeted
complexes can be internalized by virtually any cells in the body.
This nonspecific delivery of siRNA can result in serious adverse
side effects on healthy tissues and limit clinical applications of
this approach (Ikeda et al. (2006) Pharm. Res., 23:1631-1640;
Oliveira et al. (2006) J. Biomed. Biotech., 2006:63675; Kim et al.
(2007) Biotech. Prog., 23:232-237). In particular, delivery of
anticancer drugs, genes, and imaging agents specifically to primary
tumor and distant metastases requires the use of a ligand specific
to receptors that are overexpressed in cancer cells (Ikeda et al.
(2006) Pharm. Res., 23:1631-1640; Oliveira et al. (2006) J. Biomed.
Biotech., 2006:63675; Kim et al. (2007) Biotech. Prog., 23:232-237;
Taratula et al. (2009) J. Controlled Release 140:284-293; Kularatne
et al. (2010) Methods Mol Biol., 624:249-265). Previously, it has
been shown that many cancer cells overexpress receptors to
Luteinizing Hormone-Releasing Hormone (LHRH) (Dharap et al. (2003)
Pharm. Res., 20:889-896; Dharap et al. (2003) J. Controlled Release
91:61-73). A combination of anticancer drugs and LHRH peptide in
one delivery system enhanced the efficacy of chemotherapy and
decreased the adverse side effects of treatment to healthy organs
(Chandna et al. (2007) Mol. Pharm., 4:668-678; Dharap et al. (2005)
Proc. Natl. Acad. Sci., 102:12962-12967; Saad et al. (2008) J.
Control Release 130:107-114).
[0171] Herein, the development and characterization of a complex
tumor-targeted Drug Delivery System (DDS) for the simultaneous
delivery of siRNA and MRI contrast agents (SPIO) specifically to
cancer cells is provided. The ability of small (.about.5 nm) SPIO
nanoparticles was used to cooperatively form complexes of siRNA
with Polypropyleneimine Generation 5 (PPI G5) dendrimers, which are
highly branched three-dimensional polymers with defined molecular
weight and a large number of peripheral functional groups (Taratula
et al. (2009) J. Controlled Release 140:284-293). To integrate
tumor-specific targeting moiety and increase steric stability, the
formulated siRNA nanoparticles were modified with
heterobifunctional Poly(ethylene glycol) (PEG). The distal end of
PEG was coupled with a synthetic analog of LHRH decapeptide as a
targeting agent.
Methods
Materials
[0172] Polypropylenimine Tetrahexacontaamine Dendrimer Generation 5
(PPI G5), 2,4,6-Trinitrobenzenesulphonic Acid (TNBSA), oleic acid,
1-octadecene, Poly (Maleic Anhydride-alt-1-Octadecene) (PMAO,
MW=30,000-50,000 Da), Poly (Diallyldimethylammonium chloride)
(PDDA, MW 120,000 Da), microsized iron (III) oxide, Sodium Dodecyl
Sulfate (SDS), and (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic
acid) (HEPES) were obtained from Sigma-Aldrich and used without
further purification. Ethidium Bromide (EtBr) solution and
.alpha.-Maleimide-.omega.-N-hydroxysuccinimide ester Poly(ethylene
glycol) (MAL-PEG-NHS) were purchased from Promega (Madison, Wis.)
and NOF Corporation (White Plains, N.Y.), respectively. The
sequence of antisense of siRNA targeted to BCL2 mRNA (obtained from
Ambion, Austin, Tex.), was 5'-GUGAAGUCAACAUGCCUGC-dTdT-3' (sense
strand; SEQ ID NO: 4) and 5'-GCAGGCAUGUUGACUUCAC-dTdT-3' (antisense
strand; SEQ ID NO: 5). The non-targeted mock siRNA (negative
control) (5'-CCUCGGGCUGUGCUCUUUU-dTdT-3' sense strand, SEQ ID NO:
10 and 5'-AAAAGAGCACAGCCCGAGG-dTdT-3' antisense strand, SEQ ID NO:
11), 5 carboxy-fluorescein (FAM) labeled siRNA were obtained from
Applied Biosystems (Ambion, Inc., Foster City, Calif.). A synthetic
analog of LHRH, Lys6-des-Gly10-Pro9-ethylamide
(Gln-His-Trp-Ser-Tyr-DLys(DCys)-Leu-Arg-Pro-NH-Et' SEQ ID NO: 3)
peptide was synthesized by Amersham Peptide Co. (Sunnyvale, Calif.)
(Dharap et al. (2003) J. Controlled Release 91:61-73; Chandna et
al. (2007) Mol. Pharm., 4:668-678; Dharap et al. (2005) Proc. Natl.
Acad. Sci., 102:12962-12967; Saad et al. (2008) J. Control Release
130:107-114). All other chemicals were purchased from Fisher
Scientific (Fairlawn, N.J.). Cisplatin (CIS) was purchased from
Sigma (St. Louis, Mo.).
Superparamagnetic Iron Oxide (SPIO) Nanoparticles Preparation
[0173] Iron oxide nanocrystals of 5 nm in diameter were synthesized
in organic solvents at high temperature. Typically, microsized iron
oxide was mixed with oleic acid, 1-octadecene, and then heated to
320.degree. C. for a certain time to produce monodisperse (5-10%
size distribution) iron oxide nanocrystals. The size of
nanoparticles was controlled by reaction time, temperature, and the
iron oxide and oleic acid concentrations. After the reaction was
completed, the mixture was cooled and the iron oxide nanocrystals
were precipitated out of 1-octadecene by chloroform/acetone, and
then re-dispersed in chloroform. These nanocrystals were highly
crystalline and uniform but were not soluble in water due to the
hydrophobic oleic acid capping layer. For solubilization of iron
oxide nanoparticles in water, a modified method based on forming
micelles through amphiphilic polymers (PMAO) for transferring iron
oxide nanocrystals from organic solvents into water was used (Yu et
al. (2006) Nanotech., 17:4483-4487; Yang et al. (2009) Clin. Cancer
Res., 15:4722-4732). The excess of PMAO amphiphilic polymers was
removed through ultracentrifugation (600,000 g for 45 minutes). 5
mg of the PMAO modified iron oxide nanoparticles was added to 20 mL
of 10 mg/mL PDDA aqueous solution in 20 mM Tris buffer. The PDDA
was allowed to absorb for 20 min under stirring. The formed
nanoparticles were purified by the method described above and used
for further studies.
Ethidium Bromide Dye Displacement Assay
[0174] Fluorescence titration of siRNA/EtBr with the complexation
agents were performed as described above for Example 2. Binding of
the complexation agents such as the mixtures of SPIO nanoparticles
with PPI G5 dendrimer caused a displacement of bound EtBr,
resulting in a decrease in the fluorescence emission intensity.
Preparation of SPIO-PPI G5-siRNA Complexes
[0175] Prior to the cooperative complexations of siRNA with SPIO
nanoparticles and PPI G5, the stock solutions of the mixtures were
prepared by adding PPI G5 dendrimer to SPIO nanoparticle solutions
with the ratio of primary to the quaternary amines equal 5:1. The
complexes of siRNA with mixture of SPIO and PPI G5 dendrimer were
prepared at amine/phosphate ratio (N/P ratio) equal to 0.73.
Briefly, siRNA solution was mixed with HEPES buffer (5 mM, pH 7.2)
and an appropriate amount of the complexation agents was added. For
in vitro studies, the final concentration of siRNA in the solution
was 4.0 .mu.M. For in vitro and in vivo studies the final
concentrations of siRNA in the solutions were 60 .mu.M and 30
.mu.M, respectively. The samples were vortexed briefly, and the
solutions were then incubated at room temperature for 30 minutes to
ensure complex formation.
Modification of SPIO-PPI G5-siRNA Complexes with PEG and LHRH
[0176] In order to modify the SPIO-PPI G5-siRNA complexes,
NHS-PEG-MAL was reacted with primary amines on the surfaces of the
particles in 5 mM HEPES buffer (pH 7.2). The ratio of primary
amines to PEG was 10:1. The reaction was carried out for 1 hour at
room temperature. PEGylated SPIO-PPI G5-siRNA complexes were then
mixed with LHRH peptide dissolved in a HEPES buffer and incubated
overnight at 4.degree. C. The ration of PEG-MAL:LHRH in the
reaction mixture was 1:2. The resulting product was dialyzed
against deionized water using a Spectra/Pore dialysis membrane with
the molecular weight cutoff of 10,000 Da obtained from Spectrum
Laboratories, Inc. (Rancho Dominguez, Calif.).
Degree of PEGylation
[0177] The percentage of amino groups available for PEGylation as
well as the decrease in their concentration after the reaction was
determined by modified TNBSA assay (Taratula et al. (2009) J.
Controlled Release 140:284-293). Briefly, 180 .mu.L solution of
either non-modified or PEGylated SPIO-PPI G5-siRNA complexes was
mixed with 4 .mu.L of TNBSA solution (0.03M in water). Absorbance
at 420 nm was measured after 30 minutes incubation at room
temperature. All absorption measurements were performed using a
Cary-500 fluorescence spectrophotometer (Varian, Inc, Palo Alto,
Calif.). The final concentration of primary amines was calculated
using standard curves. Standard curves were prepared by plotting
the average blank corrected absorption at 420 nm reading for each
standard vs. its concentration in .mu.M.
Agarose Gel Retardation Assay
[0178] The agarose gel retardation assays were performed as
described above for Example 2. Complexation of siRNA prevented
staining of siRNA by EtBr and led to the disappearance of the siRNA
band. Therefore, the fluorescent intensity of the 21 base pair band
on the gel disappeared when siRNA was complexed with SPIO
nanoparticles and dendrimers.
Evaluation of LHRH Peptide Reaction with SPIO-PPI G5-siRNA
Complexes
[0179] Determination of the presence of LHRH peptide on the surface
of SPIO-PPI G5-siRNA complexes was performed using Bicinchoninic
Acid (BCA) protein assay (Pierce, Rockford, Ill.). The BCA method
employs the reduction of Cu.sup.+2 to Cu.sup.+1 by protein in an
alkaline medium. The combination of Bicinchoninic acid and
Cu.sup.+1 creates a purple-colored product that absorbs at 562 nm.
The amount of product formed is dependent upon the amount of
protein in the sample. The spectra of the product corresponding to
free LHRH and SPIO-PPI G5-siRNA-PEG-LHRH complexes have well
defined absorbance maximum around 560 nm corresponding to the
absorbance of the BCA/copper complex. These complexes are formed as
a result of the reaction of BCA reagent with the cuprous cation
produced from the reduction of Cu.sup.+2 to Cu.sup.+1 by the LHRH
peptide (Taratula et al. (2009) J. Controlled Release 140:284-293;
Patil et al. (2009) Biomacromolecules 10:258-266). The absorbance
maximum was absent in the assay spectra of the non-targeted
complexes that are not modified with LHRH. Briefly, 20 .mu.L of the
test solution was mixed with 200 .mu.L of working reagent and left
to react for 30 minutes at 37.degree. C. The solution then was
incubated at room temperature for 10 minutes and the absorbance was
measured at 562 nm.
Dynamic Light Scattering
[0180] Dynamic Light Scattering (DLS) studies were performed as
described above in Example 2.
Atomic Force Microscopy
[0181] In order to obtain Atomic Force Microscope (AFM) images of
formulated complexes, 5 .mu.l aliquots of SPIO-PPI G5-siRNA
solutions were deposited on a freshly cleaved mica surface. After 5
minutes of incubation, the surface was rinsed with several drops of
nanopure water (Barnstead), and dried under a flow of dry nitrogen.
AFM images were obtained using Nanoscope IIIA AFM (Digital
Instruments, Santa Barbara, Calif.) in a tapping mode, operating in
ambient air. A 125 .mu.m long rectangular silicon cantilever/tip
assembly was used with a spring constant of 40 Nm-1, resonance
frequency of 315-352 kHz, and tip radius of 5-10 nm. The applied
frequency was set on the lower side of the resonance frequency. The
image was generated by a change in amplitude of the free
oscillation of the cantilever as it interacted with the sample. The
height differences on the surface are indicated by the color code,
lighter regions indicating an increase in the height of the
complexes. The height and outer diameter of formulated complexes
were measured using the Nanoscope software.
Cell Lines
[0182] Two cancer cell lines with a different level of expression
of LHRH receptors were used. Human LHRH positive A549 lung
carcinoma cells and SKOV-3 LHRH negative ovarian cancer cells were
obtained from the ATTC (Manassas, Va., USA). In addition, A549
human lung adenocarcinoma epithelial cell line transfected with
luciferase was purchased from Xenogen Bioscience, (Cranbury, N.J.).
Cells were cultured in RPMI 1640 medium (Sigma Chemical Co., Louis,
Mo.) supplemented with 10% fetal bovine serum (Fisher Chemicals,
Fairlawn, N.J.). Cells were grown at 37.degree. C. in a humidified
atmosphere of 5% CO.sub.2 (v/v) in air. All of the experiments were
performed on the cells in exponential growth phase.
Cellular Internalization of siRNA
[0183] Cellular internalization of FAM-labeled siRNA complexes were
analyzed by fluorescence (Olympus America Inc., Melville, N.Y.) and
confocal (Leica Microsystems Inc., Bannockburn, Ill.) microscopes
as previously described (Patil et al. (2009) Biomacromolecules
10:258-266; Taratula et al. (2009) J. Controlled Release
140:284-293; Garbuzenko et al. (2009) Pharm. Res., 26:382-394). To
assess cellular internalization and localization of siRNA, ten
optical sections, known as a z-series, were scanned sequentially by
a confocal microscope along the vertical (z) axis from the top to
the bottom of the cell. Prior to the visualization, A549 and SKOV-3
cells were plated (20,000 cells/well) in 6-well tissue culture
plate. The cells were treated with different formulations for 24
hours. The concentration of siRNA was 0.25 .mu.M. After 24 hours of
treatment cells were washed three times with phosphate buffered
saline (PBS) and 1 mL of fresh medium was added to each well.
In Vitro Cytotoxicity
[0184] The cellular cytotoxicity of the formulated siRNA complexes
was assessed as described above for Example 2. Briefly, A549 cells
were separately incubated in 96-well plate with different
concentrations of the studied formulations, which resulted in a
total of seven separate series of experiments: (1) Control (fresh
media); (2) Mixture of 5 nm SPIO nanoparticles and PPI G5
dendrimers; (3) 5 nm SPIO-PPI G5-siRNA complexes; (4) 5 nm SPIO-PPI
G5-siRNA-PEG-LHRH complexes; (5) CIS; (6) Mixture of CIS and 5 nm
SPIO-PPI G5-siRNA complexes and (7) Mixture of CIS and 5 nm
SPIO-PPI G5-siRNA-PEG-LHRH complexes.
Gene Expression
[0185] Quantitative reverse transcription-polymerase chain reaction
(RT-PCR) was performed as described above in Example 2.
In Vivo Study
[0186] NCR nude mice (female, 6 weeks, 20 g) were purchased from
Taconic Farms, Inc. (Germantown, N.Y.). An animal model of human
cancer xenografts was used (Taratula et al. (2009) J. Controlled
Release 140:284-293; Chandna et al. (2007) Mol. Pharm., 4:668-678;
Saad et al. (2008) J. Control Release 130:107-114). Briefly, A549
human cancer cells transfected with luciferase (5.times.10.sup.6)
were subcutaneously transplanted into the flanks of female athymic
nu/nu mice. According to the approved institutional animal use
protocol, the tumors were measured by a caliper every other day and
their volumes were calculated as d.sup.2.times.D/2 where d and D
are the shortest and longest diameter of the tumor in mm,
respectively. When the tumor reached a mean size of 50 mm.sup.3,
mice were divided into seven groups and injected intratumorally 3
times within 10 days with 150 .mu.L of the following formulations:
(1) saline (control); (2) free non-bound LHRH; (3) free non-bound
siRNA; (4) free non-bound CIS; (5) Mixture of CIS and SPIO-PPI G5;
(6) Mixture of CIS and SPIO-PPI G5-siRNA; and (7) Mixture of CIS
and SPIO-PPI G5-siRNA-PEG-LHRH complexes. The concentrations of CIS
and siRNA in the formulations were 2.5 mg/kg and 30 .mu.M,
respectively. Changes in tumor size were monitored by real-time
bioluminescence in anesthetized animals by IVIS imaging system
(Xenogen Bioscience, Cranbury, N.J.).
Statistical Analysis
[0187] Data were analyzed as described hereinabove for Example 2.
Ten animals were used in each group of in vivo experiments.
Results
[0188] siRNA complexes were prepared with a mixture of SPIO
nanoparticles and PPI G5 dendrimers, which introduced functional
primary amino groups on the surfaces of formulated siRNA complexes
for their further modification (FIG. 24). PPI dendrimers were
covered with PEG polymers and LHRH peptide as a targeting moiety
specific to cancer cells was conjugated to the distal end of the
polymer. The efficiency of 5 nm and 10 nm SPIO nanoparticles to
cooperatively provoke siRNA complexation with PPI G5 was studied by
ethidium bromide dye displacement assay. The ratio of free amines
(nitrogen) on PPI dendrimers and SPIO nanoparticles to phosphate on
the siRNA (N/P ratio) was employed to quantify the efficacy of
cooperative siRNA condensation. Quantitative analysis of the
mixture's complexation efficiency reveals that the mixture of 5 nm
SPIO and PPI G5 is more effective in provoking siRNA complexation
(the apparent end point of complexation of N/P ratio=0.73) than PPI
G5 dendrimer alone (N/P ratio=1.13) and 10 nm SPIO with PPI G5 (N/P
ratio=1.3) (FIG. 25A, end points of complexation are denoted as
circles in the insert). Therefore, the mixture of 5 nm SPIO and PPI
G5 was the most effective complexation agent and was employed for
the development of multifunctional nanomedicine platform for cancer
specific delivery of siRNA. Agarose gel retardation assay was
additionally involved to confirm the formation of 5 nm SPIO-PPI
G5-siRNA complexes formed at N/P ratios which represented the
apparent complexation end points obtained from ethidium bromide dye
displacement assay. It was found that a complete binding of siRNA
(without the presence of a trailing band) with the mixtures of 5 nm
SPIO and PPI G5 dendrimers was observed in comparison to free siRNA
(FIG. 25B).
[0189] DLS measurements at a 108.degree. scattering angle were used
to estimate the apparent hydrodynamic diameters of the resulting
siRNA complexes. The results of DLS measurements demonstrate that
the average diameter of 5 nm SPIO-PPI G5-siRNA complexes was
169.8.+-.28.4 nm (FIG. 26A). In addition to DLS measurement, the
formation of nanosized siRNA complexes has further been confirmed
by AFM (FIG. 26B). AFM analysis verified that 5 nm SPIO
nanoparticles cooperatively with PPI G5 dendrimers could
effectively produce complexes with siRNA leading to the formation
of discrete particles with an average diameter of 214.3.+-.53.1 nm.
The differences in the size of nanoparticles probably reflect the
differences in a sample preparation for the size measurements by
two different methods. DLS was performed on nanoparticles in a
fully hydrated state in solution, whereas AFM studies were carried
out on samples dried to the mica surface, which resulted in
flattering of the nanoparticles on the mica surface during the
drying process (Wang et al. (2008) Clin. Cancer Res.,
14:3607-3616).
[0190] The PEGylation of SPIO-PPI G5-siRNA complexes was carried
out by coupling of linear MAL-PEG-NHS to the amino groups on the
surface of the complexes, which were introduced by PPI G5
dendrimers. The availability of the primary amines in the structure
of the prepared siRNA complexes before PEGylation as well as the
decrease in their concentration after PEGylation has been estimated
by the TNBSA assay. The result reveals that the degree of
PEGylation was 70% for SPIO-PPI G5-siRNA complexes.
[0191] To examine the influence of nanoparticles coating on their
cellular uptake, PEGylated and non-PEGylated siRNA complexes were
incubated with A549 cancer cells in a fresh medium. Fluorescence
microscopy studies revealed the fact that PEG modification of
SPIO-PPI G5-siRNA complexes enhance their sterical stability and
prevent the aggregation of complexes that was abundant in
non-PEGylated complexes (FIG. 27, compare panels A and B). On the
other hand, non-PEGylated complexes provided for an effective
delivery of labeled siRNA into the cells (FIG. 27A). As expected,
PEGylation of the siRNA complexes decreased their internalization
by cancer cells.
[0192] In order to evaluate the biological activity of the
delivered siRNA, the siRNA targeted to BCL2 mRNA was used in the
present study. FIG. 28 shows the expression of the BCL2 gene in
A549 and SKOV-3 human cancer cells treated with siRNA delivered by
different SPIO-PPI G5 complexes. The suppression of BCL2 mRNA by
the PEGylated complexes was substantially lower when compared with
the corresponding non-PEGylated system (lines 2 and 3). The
sufficient decrease in gene silencing activity of the PEGylated
complexes corroborate with cellular internalization data. To
exclude nonspecific effects on gene expression by SPIO-PPI G5
complexes alone without bound siRNA, whether the mixtures of SPIO
nanoparticles with dendrimers could impact the BCL2 gene expression
was examined. RT-PCR analysis demonstrated that the employed siRNA
delivery systems did not induce statistically significant changes
in the expression of BCL2 mRNA in A549 cancer cells at the studied
concentrations (FIG. 28, line 5). Similarly, a mocked siRNA duplex
with a scrambled sequence having no significant homology to any
known gene sequences was used in this series of the experiments as
a negative control. RT-PCR data demonstrated that complexes with
such mocked siRNA did not show any statistically significant
inhibition of BCL2 mRNA expression confirming the specificity of
BCL2 mRNA functional knock-down (FIG. 28, line 6).
[0193] In order to conjugate a targeting moiety (LHRH decapeptide)
to the siRNA nanoparticles, the maleimide group at the distal end
of the PEG-chain was coupled to thiol group presented by cysteine
residue in modified LHRH sequence. The presence of LHRH peptide on
the complex surface was confirmed by Bicinchoninic Acid (BCA)
protein assay (Thermo Fisher Scientific Inc., Rockford, Ill.)
according to manufacture protocol. As shown in FIG. 26A, DLS
measurements reveal that the diameter of modified SPIO-PPI G5-siRNA
complexes was 212.0.+-.35.6 nm, respectively. The increase in the
size of the modified siRNA complexes compared to nonmodified ones
could be explained by the presence of the polymer layer on the
surface of siRNA complexes.
[0194] In vitro studies were performed to characterize the
influence of LHRH peptide as a targeting moiety on the uptake and
intracellular activity of the entrapped siRNA. The fluorescence
microscopy images demonstrated a sufficient increase in the
intracellular internalization of LHRH-targeted complexes by A549
cancer cells which overexpress LHRH receptors (FIG. 27C, D). In
contrast, cellular uptake of tumor-targeted siRNA complex in LHRH
negative SKOV-3 cells was substantially less when compared with
non-targeted complexes (FIG. 27D). These experiments confirmed that
the targeted shielded nanoparticles indeed delivered the siRNA
specifically to the cancer cells, which overexpress the targeting
receptors.
[0195] Theoretically, the formulated siRNA complexes could adhere
to the surface of LHRH-positive cancer cells and erroneously be
visualized on microscopic images as internalized within the cell.
To exclude such errors, the distribution of LHRH targeted siRNA
complexes was analyzed in different cellular layers from the upper
to the lower surfaces of the cell using confocal fluorescent
microscopy. In these experiments, the formulated complexes with FAM
labeled siRNA were incubated with human A549 cancer cells. The
cells were subjected to confocal microscopy. The z-section of
single cells transfected with the modified complexes, formed by
complexation of siRNA with the mixture of SPIO nanoparticles and
PPI G5 dendrimer, and showed their homogeneous and uniform
distribution in all layers of the cell from the top surface to the
bottom (FIG. 29). To assess the ability of LHRH targeted complexes
not only to deliver siRNA but knockdown targeted gene expression,
complexes with BCL2-specific siRNA were prepared. In this series of
the experiments, both LHRH positive (A549) and negative (SKOV-3)
cancer cells were treated with the prepared siRNA complexes for 24
hours. The RT-PCR data obtained revealed that the LHRH modification
of siRNA complexes restore the knockdown activity for siRNA
complexes, which was decreased after PEGylation (FIG. 28, line 4).
On the hand, the silencing effect of the BCL2-targeted siRNA was
not significant in LHRH-negative cancer cells (FIG. 28, line 7),
which is in good agreement with the siRNA cellular internalization
result represented in FIG. 27. Therefore, as expected, LHRH peptide
proved its capability to target effectively the siRNA complexes to
the specific receptors in the plasma membrane of cancer cells.
[0196] The influence of the formulated siRNA delivery systems on
cell viability was investigated in A549 human lung cancer cell line
by the MTT assay. FIG. 30A shows the average data from three
different experiments with increasing concentration of the
complexes. One can see that over 95% average cell viability was
observed for both 5 nm SPIO-PPI G5 and 5 nm SPIO-PPI G5-siRNA
delivery systems at the concentrations used for in vitro and in
vivo experiments of the present study. At a maximum available
concentration, the mean cell viabilities for the targeted SPIO-PPI
G5-PEG-LHRH complex was 85% compared with that of the control,
respectively.
[0197] The ability of the developed siRNA delivery system to
enhance efficiency of a chemotherapeutic drug such as CIS was
evaluated in the current study both in vitro and in vivo. The
cellular cytotoxicity of Cisplatin alone and in combination with
non-targeted SPIO-PPI G5-siRNA or targeted SPIO-PPI
G5-siRNA-PEG-LHRH delivery systems was assessed using a modified
MTT assay. Data in FIG. 30B shows that cytotoxicity of CIS against
multidrug resistant human cancer cells was sufficiently enhanced in
the presence of non-targeted or LHRH targeted siRNA delivery
vectors. The maximum enhancement of anticancer activity of CIS was
observed at the lower concentration range of the drug (1
.mu.g/mL-150 .mu.g/mL).
[0198] Antitumor activities of the proposed formulations with
corresponding controls were studied in vivo using subcutaneous
xenograft model of human cancer. The progression of tumor growth
was monitored by an IVIS imaging system and by measuring the tumor
volume (FIG. 31, upper and bottom panels, respectively). It was
found that free LHRH and non-conjugated naked siRNA did not
significantly influence the growth of the tumor (FIG. 31, bottom
panel, curves 2-3). Free CIS limited the growth of the tumor at the
last day of the treatment on 36.2% when compared with untreated
control (FIG. 31, image 4, curve 4). Simultaneous delivery of CIS
and SPIO-PPI-G5 dendrimer complex slightly increased the antitumor
activity of the drug (FIG. 31, image 5, curve 5). The suppression
of cellular antiapoptotic defense by siRNA targeted to BCL2 mRNA,
delivered by SPIO-PPI G5-siRNA complex simultaneously with CIS
significantly enhanced the antitumor activity of the drug. In fact,
the tumor volume decreased on 67.5% after the combinatorial
treatment when compared with untreated control (FIG. 31, image 6,
curve 6). Targeting of siRNA complexes specifically to the tumor by
LHRH peptide led to the further enhancement of the antitumor
activity of CIS. The tumor volume decreased on 75.5% when compared
with untreated control (FIG. 31, image 7, curve 7).
[0199] Multifunctional nonviral vector have been for siRNA delivery
based on SPIO nanoparticles modified with PDDA and PMAO, which
contain quaternary ammonium and carboxylic functional groups on the
periphery for siRNA condensation and endosomal release. These SPIO
nanoparticles demonstrated high efficiency to form complexes with
siRNA and to facilitate their internalization by the cancer cells.
Cellular uptake of such SPIO-siRNA complexes most probably occurred
by adsorptive-mediated endocytosis, which is triggered by
electrostatic interactions between the negatively charged plasma
membrane and the positively charged complexes. Targeting of the
SPIO-siRNA complexes specifically to cancer cells by incorporating
a ligand to the receptors overexpressed in the plasma membrane of
cancer cells can offer at least three advantages. First, it
switches the mechanism of cellular internalization toward more
efficient receptor-mediated endocytosis. Second, specific targeting
to cancer cells prevents rapid clearance of the siRNA cationic
complexes by liver, spleen, and kidney after systemic
administration (Taratula et al. (2009) J. Controlled Release
140:284-293; Fischer et al. (2004) Drug Metab. Disposit.,
32:983-992). Third, the delivery of therapeutic payload
specifically to cancer cells limits adverse side effects of the
treatment on healthy organs by changing its organ distribution
toward the preferential accumulation in the tumors (Taratula et al.
(2009) J. Controlled Release 140:284-293; Chandna et al. (2007)
Mol. Pharm., 4:668-678; Dharap et al. (2005) Proc. Natl. Acad.
Sci., 102:12962-12967). Consequently, in the present study,
tumor-targeted superparamagnetic iron oxide nanoparticles-dendrimer
complexes have been characterized for simultaneous delivery
specifically to tumor cells of siRNA and MRI-contrast agents.
Therefore, the proposed complex multifunctional drug delivery
platform can be used for simultaneous suppression of cellular
resistance by siRNA and MRI imaging of the system itself, and
primary tumor or metastases. Experimental data show the following
advantages of the proposed delivery system.
[0200] It is well-known that siRNA complexes should have an optimal
size and proper shape for effective gene delivery because that
often governs the transfection efficiency, cytotoxicity, and tissue
targeting of an entire system in vivo (Fischer et al. (2004) Drug
Metab. Disposit., 32:983-992). Generally, in order to enable its
effective penetration into tissue, the size of gene delivery
vehicles should not exceed 250 nm (Wood et al. (2005) Angewandte
Chemie-Intl. Ed., 44:6704-6708), although the optimal size of the
particles is still under debate. Direct measurements by several
independent approaches determined that the size of complexes
developed in the present study was approximately 200 nm (with
complexated siRNA). The nanoparticles were compact and close to
spherical shape. Consequently, one could expect that such
dendrimer-based systems will provide for an efficient delivery of
its payload into cancer cells (Patil et al. (2009)
Biomacromolecules 10:258-266; Taratula et al. (2009) J. Controlled
Release 140:284-293; Patil et al. (2008) Bioconjugate Chem.,
19:1396-1403). Further investigations confirmed this
suggestion.
[0201] Cytotoxicity of gene transfection vectors including viral
vectors, inorganic nanoparticles, cationic liposomes, and polymeric
cations is a major barrier for their efficient use for the delivery
of therapeutic genes (Bessis et al. (2004) Gene Ther., 11:S10-S17).
Recently, it has been demonstrated that PPI dendrimers can
intrinsically alter the expression of many endogenous genes, the
nature and extent of which were dependent on the dendrimer
generation, and cell type (Omidi et al. (2005) J. Drug Targeting
13:431-443). Although cytotoxicity of a nanocarrier itself is not
an issue for the delivery of anticancer drugs with much more higher
cytotoxicity, it was found that the proposed targeted and
nontargeted SPIO-PPI G5 vehicles alone and in combination with
siRNA possessed low cytotoxicity. Moreover, a mixture of SPIO with
PPI G5 dendrimers alone without siRNA did not influence the
expression of the targeted BCL2 gene. Such low toxicity of the
modified siRNA complexes makes them attractive for in vivo delivery
of nontoxic compounds. Consequently, similar drug delivery systems
can be used for applications other than cancer chemotherapy.
[0202] It is known that siRNA complexes are usually easily
opsonized and removed from the circulation long prior to completion
of their main function (de Wolf et al. (2007) Intl. J. Pharm.,
331:167-175; Dash et al. (1999) Gene Ther., 6:643-650). Chemical
modification of siRNA delivery vector with certain synthetic
polymers, such as PEG, is the most frequent way to increase the in
vivo longevity in the systemic circulation of siRNA delivery
vectors. The layer of hydrophilic polymer (in most cases PEG)
sterically hinders interactions of blood components with the
positively charged surface of siRNA complexes and enhances their
stability in the blood stream (Schiffelers et al. (2004) Nuc. Acids
Res., 32:e149; Mao et al. (2006) Bioconjugate Chem., 17:1209-1218).
However, simultaneously while improving the pharmacokinetics,
PEGylation usually limits cellular internalization in vivo (stealth
effect) (Taratula et al. (2009) J. Controlled Release 140:284-293).
It is known that neutral surface charge of PEGylated siRNA
complexes limits their interactions with a negatively charged cell
membrane when compared with positively charged non-modified siRNA
complexes. To overcome these obstacles, the modification of
sterically stabilized siRNA delivery carriers with cell targeting
ligands is usually used in order to enhance its transfection
activity. The different targeting moieties including, galactose,
folate, RGD-peptide and antibodies were examined for the delivery
of DNA and siRNA. Recently, it was found that the receptor for LHRH
is overexpressed in many types of human cancer cells and was not
detectably expressed in healthy human visceral organs (Dharap et
al. (2003) Pharm. Res., 20:889-896; Dharap et al. (2003) J.
Controlled Release 91:61-73). Furthermore, it has been shown that
the use of the LHRH peptide for the targeting of a polymeric
anticancer drug delivery system to cancer cells substantially
limits its adverse side effects on healthy tissues and
significantly enhances the antitumor efficacy of the anticancer
drug. Therefore, based on these results the synthetic analog of
LHRH peptide was selected as a targeting moiety to enhance the
internalization of the developed siRNA delivery system specifically
by cancer cells. The results of the in vitro and in vivo
experiments of this tumor-targeted system showed that an
incorporation of LHRH peptide substantially improved cellular
internalization of siRNA, increased its transfection efficiency,
and enhanced the antitumor activity of drug.
[0203] The data clearly show that the combinatorial delivery of
siRNA with anticancer drug substantially enhanced the efficiency of
chemotherapy leading to the more significant limitation of the
tumor growth. Therefore, it is important to deliver siRNA inside
tumor cells simultaneously with an anticancer drug. The delivery of
siRNA requires an appropriate carrier because naked siRNA is
unstable in the blood stream and poorly penetrates cells. The
proposed in the present research delivery system significantly
improves the stability of siRNA in plasma and provides for its
efficient cellular internalization. In addition, an incorporation
of a tumor-targeting moiety (LHRH peptide) into the DDS permits the
delivery of siRNA specifically into tumor cells further enhancing
antitumor effects of the drug and limiting adverse side effects of
the treatment on healthy organs.
[0204] In summary, the designed siRNA delivery vector based on SPIO
nanoparticles modified with PPI G5 dendrimers and PEG combines the
cell-targeted selectivity with the specificity of siRNA. The
modular chemical design of the proposed system allows for the
substitution of used cancer targeting moiety with other ligands, or
combinations of ligands, to selectively target other type of cancer
cells.
[0205] A number of publications and patent documents are cited
throughout the foregoing specification in order to describe the
state of the art to which this invention pertains. The entire
disclosure of each of these citations is incorporated by reference
herein.
[0206] While certain of the preferred embodiments of the present
invention have been described and specifically exemplified above,
it is not intended that the invention be limited to such
embodiments. Various modifications may be made thereto without
departing from the scope and spirit of the present invention, as
set forth in the following claims.
Sequence CWU 1
1
11124DNAArtificial SequenceSynthetic sequence 1ttcaagatcc
atcccgacct cgcg 24220DNAArtificial SequenceSynthetic sequence
2cagcgtgcgc catccttccc 2039PRTArtificial SequenceSynthetic sequence
3Gln His Trp Ser Tyr Lys Leu Arg Pro1 5 421DNAArtificial
SequenceSynthetic sequence 4gugaagucaa caugccugct t
21521DNAArtificial SequenceSynthetic sequence 5gcaggcaugu
ugacuucact t 21620DNAArtificial SequencePrimer 6ggattgtggc
cttctttgag 20720DNAArtificial SequencePrimer 7ccaaactgag cagagtcttc
20820DNAArtificial SequencePrimer 8acccccactg aaaaagatga
20920DNAArtificial SequencePrimer 9atcttcaaac ctccatgatg
201021DNAArtificial SequenceSynthetic sequence 10ccucgggcug
ugcucuuuut t 211121DNAArtificial SequenceSynthetic sequence
11aaaagagcac agcccgaggt t 21
* * * * *